Anti-tenascin C antibodies and uses thereof

ABSTRACT

There is provided antibodies or antigen-binding fragments, derivatives or variants thereof which are capable of binding to the FBG domain of tenascin-C. There are also provided uses of such antibodies or antigen-binding fragments, derivatives or variants thereof, as well as methods of identifying such antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of, and claims priority to,U.S. patent application Ser. No. 16/104,610, filed Aug. 17, 2018, nowallowed, which is a continuation of U.S. patent application Ser. No.15/501,979 filed Feb. 6, 2017, now U.S. Pat. No. 10,093,723, which wasfiled under 35 U.S.C. § 371 as the U.S. national phase of InternationalApplication No. PCT/GB2015/052298, filed Aug. 7, 2015, which designatedthe U.S. and claims the benefit of priority to GB 1414021.4, filed Aug.7, 2014, each of which are hereby incorporated herein in their entiretyincluding all tables, figures and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 8, 2019, isnamed 314641-00043_SequenceListing.txt and is 144 kilobytes in size.

The present invention relates to antibodies for binding thefibrinogen-like globe (FBG) domain of tenascin-C and their use in thediagnosis, determination of prognosis and/or treatment of disordersassociated with chronic inflammation, as well as methods of identifyingsuch antibodies.

Inflammation is the complex biological response of tissues to harmfulstimuli, such as pathogens, tissue damage, or irritants. It is aprotective attempt by the tissue to remove the injurious stimuli as wellas initiate the healing process for the tissue. Abnormalities associatedwith inflammation comprise a large, unrelated group of disorders whichunderlie a variety of human diseases (inflammatory disorders). Examplesof diseases with an inflammatory aspect include (but are not limited to)asthma, autoimmune disease, glomerulonephritis, allergy(hypersensitivities), cancer, inflammatory bowel diseases, reperfusioninjury, rheumatoid arthritis and transplant rejection.

In particular, chronic inflammation is a debilitating and seriouscondition associated with many of the above diseases and ischaracterised by persistent inflammation at a site of infection orinjury, or persistent inflammation of an unknown origin, or in relationto altered immune responses such as in autoimmune disease.

Rheumatoid arthritis (RA) is a typical example of, though by no meansthe only, a chronic inflammatory condition. RA is characterised bysynovial inflammation and destruction of joint cartilage and bonemediated by persistent synthesis of pro-inflammatory cytokines andmatrix metalloproteinases (MMPs). Biological compounds that suppress thesynthesis of inflammatory cytokines such as TNFα and IL-6 are successfulat treating RA in the short-term. However, repeated treatments arerequired, which renders this an expensive therapeutic approach, and doesnot provide long-term remission. Furthermore, total systemic suppressionof cytokine function is not without inherent problems such as increasedinfectious risk. Thus, despite advances in care, there remains an unmetneed for an economical mode of treatment of chronic inflammatoryconditions that is efficacious over the long term (Smolen (2006) andWilliams (2007)).

The mechanisms that underpin disease chronicity remain unclear and thefactor(s) that drive the prolonged expression of inflammatory anddestructive mediators are currently unknown.

Toll-like receptors (TLRs) play a key role in driving the production ofinflammatory mediators in RA and blockade of TLR function may be ofsignificant clinical benefit (reviewed in Brentano (2005) and O'Neill(2002)). This receptor family forms an integral part of the immunesystem. TLRs mediate host defence against infection and injury byrecognising both pathogen-associated molecular patterns (PAMPs) anddamage-associated molecular patterns (DAMPs) (Matzinger (2002)). DAMPsare endogenous pro-inflammatory molecules generated upon tissue injuryand include intracellular molecules released from damaged or necroticcells, fragments of extracellular matrix (ECM) molecules or ECMmolecules up regulated upon injury (reviewed in Bianchi (2007) andGordon (2002)).

Upon activation, TLRs promote both innate and adaptive immune responsesincluding stimulation of expression of pro-inflammatory cytokines andMMPs (Medzhitov (2002)). TLRs are expressed at high levels in synovialtissue from RA patients (Radstake (2004), Roelofs (2005), Sacre (2007),and (Sacre, manuscript submitted 2008) and mice with targeted deletionsor loss of function mutations in TLR4 are protected from experimentalarthritis (Choe (2003) and Lee (2005). Furthermore, inhibitors of TLR4can reduce destructive arthritis in mice (Abdollahi-Roodsaz (2007)) anda putative TLR4 inhibitor improved symptoms in 15 out of 23 patientswith moderate to severe RA in a preliminary phase I trial (Vanags(2006). However, it is unclear which TLR ligand(s) are involved indisease pathogenesis.

Tenascin-C (TNC) is an ECM glycoprotein that is associated with tissueinjury and wound repair. Tenascin-C is expressed specifically duringactive tissue remodelling during embryogenesis, being first observedduring gastrulation and somite formation. In later stages of developmentexpression is restricted to sites of branching morphogenesis of mammarygland and the lung, in the developing skeleton, cardiovascular systemand in connective tissues at sites of epithelial to mesenchymaltransformation. Expression is down-regulated once these processes ceaseand before embryogenesis is complete (Jones (2000)).

Tenascin-C is not normally expressed in healthy adult tissue but, inadults, is specifically and transiently up-regulated during acuteinflammation and persistently expressed in chronic inflammation(reviewed in Chiquet-Ehrismann (2003)). Immunohistochemical studies showthat little tenascin-C is expressed in normal human joints but levelsare greatly increased in RA synovia, in areas of inflammation andfibrosis, specifically below the synovial lining, in the invading pannusand around blood vessels (Cutolo (1992), MacCachren (1992) and Salter(1993)). There is also a significant increase in tenascin-C levels insynovial fluid from RA patients (Chevalier (1994) and Hasegawa (2007))and in RA cartilage (Salter (1993) and Chevalier (1994)).

Tenascin-C is a large hexameric protein of 1.5 million Da. Each chaincomprises different domains, including an assembly domain (TA), EGF-likerepeats (EGF-L), fibronectin type III-like repeats (TNIII) and afibrinogen-like globe (FBG) (reviewed in Orend (2005)). The sequences oftenascin-C and its domains are shown in FIG. 1.

The inventors have shown previously that tenascin-C is apro-inflammatory stimulus and that it is required for destructive jointinflammation observed in arthritis and is involved in the prolonging ofthe inflammatory response characterising the chronic inflammatorycondition. In particular, tenascin-C has been shown to be an endogenousactivator of TLR4 and it has been demonstrated that this molecule isrequired for destructive joint inflammation (WO 2010/103289).

In WO 2010/103289, a role for tenascin-C in mediating an immune responsein the joint was demonstrated by induction of joint inflammation uponintra-articular injection of the FBG domain of tenascin-C in mice invivo. Moreover, acute joint inflammation induced by zymosan was not asprolonged in tenascin-C deficient mice. Both the wild type andtenascin-C null mice responded to acute inflammation induction byzymosan equally, demonstrating that tenascin-C does not appear to beinvolved in the initiation of inflammation. However, the less persistentsynovitis exhibited by tenascin-C null mice indicates a role in themaintenance of joint inflammation. The importance of tenascin-C inprolonging joint inflammation was underscored by the observation thattargeted deletion of tenascin-C protected mice from sustained erosivejoint inflammation during arthritis induced by immunization with mBSA.

Tenascin-C was shown to be capable of activating cells in the joint andthe primary active domain of tenascin-C has been mapped to thefibrinogen-like globe (FBG), a 227 amino acid (26.9 kDa) globular domainat the C terminal of the molecule (Siri (1991)).

Addition of FBG to synovial membrane cultures from RA patients enhancedthe spontaneous release of pro-inflammatory cytokines. It alsostimulated synthesis of TNF-α, IL-6 and IL-8 in primary humanmacrophages and IL-6 in RA synovial fibroblasts via activation of TLR4and MyD88 dependent signalling pathways.

It has been shown that, as in the case of LPS, TLR4 expression isnecessary for induction of cytokine synthesis by FBG. However, unlikeLPS, neither CD14 nor MD-2 appears to be required for TLR-4 activation.CD14 is dispensable for activation of TLR4 by other ligands. It is notrequired for TLR4 to respond to lipid A in a MyD88 dependent manner(Jiang (2005)), fibronectin EDA (extra domain A) can activate mast cellseven in the absence of CD14 (Gondokaryono (2007)) and hyaluronic acidactivation of human monocytic THP-1 cells requires a complex of TLR4,CD44 and MD-2, but not CD14 (Taylor (2007)).

Formation of distinct receptor complexes by each TLR4 ligand mayfacilitate recruitment of different intracellular adapter/signallingmolecules. This may account for the differential cellular responses weobserve with FBG and LPS. Similarly, hyaluronic acid activation of theTLR4 and CD44 complex induces a pattern of gene expression in mousealveolar macrophage cell lines that is different to LPS (Taylor (2007)).

The tightly regulated pattern of expression of tenascin-C makes it anattractive target for treating chronic inflammation. It is predominantlyabsent from healthy adults, however expression is specifically inducedupon tissue injury. During acute inflammation tenascin-C is transientlyexpressed: induction often precedes inflammation and both mRNA andprotein are absent from the tissue by the time inflammation is resolved(reviewed in Chiquet-Ehrismann (2003)).

Persistent expression of tenascin-C has now been shown to be associatedwith chronic inflammation. In addition to RA, increased tenascin-Clevels are observed in other autoimmune diseases including multiplesclerosis (Gutowski (1999)) and Sjogrens disease (Amin (2001)), and innon-healing wounds and diabetic and venous ulcers (Loots (1998)). Denovo synthesis of tenascin-C correlates well with the intensity ofinflammation in diseases of the oral mucosa and plasma levels oftenascin-C are a reliable indicator for the activity of inflammatorybowel diseases before and after medication or surgery (reviewed inChiquet-Ehrismann (2003)).

WO 2010/103289 describes the use of agents for modulation of a chronicinflammatory response wherein the agent modulates the biologicalactivity of tenascin-C and their use in treating conditions associatedwith chronic inflammation. However, there remains an ongoing need fornew and improved treatments for such conditions.

Clark et al. (1997) (52) describes investigations into tenascin anddescribes an antibody specific for the FBG domain. That antibody is ofmouse origin, and therefore is not suitable as a therapeutic.Furthermore, that antibody is described only as having the property ofinterfering with “lymphocyte rolling”, which is believed to be a measureof cell migration, and not cell activation and production ofinflammatory cytokines. The cellular counter receptor involved in thecell rolling activity described by Clark was not identified.Participation in the process of cell rolling or migration is notbelieved to be a significant property of TLR4, nor was TLR4 included byClark in a list of potential candidate counter receptors involved inrolling behaviour. Additionally, not all antibodies which bind FBG areable to inhibit the production of inflammatory cytokines. The novel setof improved properties of the antibody sequences described herein wasneither taught nor suggested by Clark, nor were they tested for. Thus,there is nothing to indicate that the antibody described in Clark bindsto the same region of FBG as the antibodies described herein, it appearsnot to be related to TLR4 activity since TLR4 is not commonly considered(including by Clark by its omission from their list of potentialcandidates) to be involved in lymphocyte rolling activity, the functionstudied by Clark et al is unrelated to the key anti-inflammatoryproperty of the novel antibodies described herein.

Therefore, there remains a need to produce new and improved antibodiesspecific for the FBG domain of tenascin-C, particularly those which havethe properties required to make them useful as therapeutics.

The inventors have designed antibodies and fragments thereof withproperties that are suitable for use in therapy, in particular humanantibodies, with very high affinity to the fibrinogen-like globe (FBG)domain of tenascin-C, and which neutralise the biological activity ofFBG. These high affinity antibodies are useful in a variety oftherapeutic methods, such as those which use anti-FBG antibody moleculesin the diagnosis or treatment of tenascin-C related disorders,particularly those associated with chronic inflammation, includingrheumatoid arthritis (RA). The antibodies are also useful in relateddiagnostic and prognostic methods.

In a first aspect of the invention there is provided an antibody orantigen-binding fragment, derivative or variant thereof which is capableof binding to the FBG domain of tenascin-C, wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises: oneor more sequences selected from SEQ ID NOs: 9-15, 5, 125, 36, 37, 30-35,38-47, 115-118 and 140; and/or one or more sequences selected from SEQID NOs: 1-8, 124, 48-91, 128-138, 112-114 and 139; and/or one or moresequences selected from SEQ ID NOs: 5, 13, 16-21, 126, 119-121 and 141;and/or one or more sequences selected from SEQ ID NOs: 22-29, 127,122-123 and 142.

The sequence ID numbers (SEQ ID NOs) refer to those designating theparticular antibody and antibody related sequences listed in Examples 9and 11. Where not explicitly stated, reference to “antibodies” includesantigen-binding fragments, derivatives or variants thereof.

In one embodiment the antibody or antigen-binding fragment, derivativeor variant thereof according to the present disclosure comprises a heavychain variable region and/or a light chain variable region which ishuman or humanised.

In one embodiment the antibody or antigen-binding fragment, derivativeor variant thereof according to the present disclosure is human orhumanised, including fully human.

In one embodiment the antibody or antigen-binding fragment, derivativeor variant thereof according to the present disclosure neutralises,reduces or blocks activation of TLR4.

In one embodiment the antibody or antigen-binding fragment, derivativeor variant thereof according to the present disclosure comprises heavychain variable region and/or a light chain variable region which ishuman or humanised (including a fully human antibody or bindingfragment), which inhibits release for a cytokine such as IL-8 in anassay described herein.

In one embodiment the antibody or antigen-binding fragment, derivativeor variant thereof according to the present disclosure is specific tothe FBG domain of tenascin C. That is to say it does not cross-reactwith other members of the tenascin-C family.

In one embodiment the antibody or antigen-binding fragment, derivativeor variant thereof according to the present disclosure is cross-reactivewith the FBG domain of at least murine and/or rattus tenascin C, inparticular cross-reactive with the FBG domain of primitive tenascin-C.This is particularly important for a potential therapeutic as it allows,safety and efficacy studies to be performed in vivo beforeadministration to humans.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof according to the presentdisclosure which comprises 6 CDRs, namely 3 heavy chain CDRs and 3 lightchains CDRs, for example disclosed in an antibody herein and furthercomprising a human framework.

In one embodiment the antibody or antigen-binding fragment, derivativeor variant thereof is provided with an affinity for the FBG domain ofhuman tenascin C of at least 100 nM or higher, for example 50 nM orhigher, such as 1 nM or higher affinity. The higher the affinity thelower the numerical value.

In one embodiment the present disclosure extends to an “antibody”explicitly disclosed in the sequence listing provided in Examples 9 or11.

In one embodiment there is provided an antibody, for example a human orhumanised antibody, which cross-blocks an antibody disclosed herein orcompetively binds the same epitope as an antibody disclosed herein.

In one embodiment the antibody of the present disclosure is provided ina full length antibody format, for example a format with no-effectorfunction such as an IgG4 format.

In one embodiment the antibody or antigen-binding fragment, derivativeor variant thereof is for use in therapy, in particular use in thetreatment of an autoimmune disease or inflammation, in particular acondition describe herein.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may therefore be an antigen-bindingfragment. The antigen-binding “fragment” may be selected from the groupconsisting of Fv fragments (e.g. single chain Fv and disulphide-bondedFv), Fab-like fragments (e.g. Fab fragments, Fab′ fragments and F(ab)₂fragments), single variable domains (e.g. V_(H) and V_(L) domains) anddomain antibodies (dAbs, including single and dual formats [i.e.dAb-linker-dAb]).

The terms antibody “derivative” and “variant” refer to any modifiedantibody molecule (including homologues) that is capable of binding toan antigen in an immunoassay format that is known to those skilled inthe art, such as a fragment of an antibody (e.g. Fab or Fv fragment), ora modified antibody molecule that is modified by the addition of one ormore amino acids or other molecules to facilitate coupling theantibodies to another peptide or polypeptide, to a large carrier proteinor to a solid support (e.g. the amino acids tyrosine, lysine, glutamicacid, aspartic acid, cysteine and derivatives thereof, NH2-acetyl groupsor COOH-terminal amido groups, amongst others).

The variant may include a variation of the amino acid sequence of theantibody. For example, the amino acid sequence of the variant might be50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to the specified antibody at the amino acid sequence level.

The variant may also include sequence changes in order to utilise themost common natural human germline antibody frameworks and CDRdiversity. This may be done by tailor-engineering of key residues withamino acids commonly found in natural human antibodies. This approachminimizes the likelihood of anti-antibody reactions in humans, sincegermline antibody framework sequences are highly tolerated betweenindividuals. This technique is known as “germlining” and the resultantsequences are termed “germlined sequences”. Sequences may be fully orpartially germlined. Examples of germlined sequences of the antibodiesof the invention are described in Example 11.

The antibody of the invention may bind to the FBG domain that bindsTLR4, blocking TLR4 activation directly, for example by physicallyoccluding the binding site with TLR4. Whilst not wishing to be bound bytheory the present inventors have evidence to suggest the TLR4 binds anFBG domain of tenascin C directly. This has not been previouslyestablished even though tenascin C was known to be capable of enhancingthe activity of TLR4.

Alternatively the antibody may bind to a part of the FBG domain thatdoes not bind TLR4, but this may still prevent TLR4 activation(allosteric effect); the antibody may bind to an FBG domain thatinteracts with another receptor blocking its activity, this may or maynot have an impact on TLR4 activity; and/or the antibody may bind to theFBG domain which does not bind to any other receptor but still preventsthe activation of this receptor (allosteric effect).

In one embodiment the disclosure relates to an antibody orantigen-binding fragment, derivative or variant thereof that binds theFBG domain but does not inhibit activation of TLR4 and/or release ofcytokines, in particular those described herein, such as IL-8.

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFvand dAb antibody fragments can be expressed in and secreted from E.coli, thus allowing the facile production of large amounts of the saidfragments.

Also included within the scope of the invention are modified versions ofantibodies and antigen-binding fragments thereof, e.g. modified by thecovalent attachment of polyethylene glycol or other suitable polymer.Conjugates of antibodies and antigen-binding fragments thereof are alsoincluded, e.g. antibody-drug conjugates.

Methods of generating antibodies and antibody fragments are well knownin the art. For example, antibodies may be generated via any one ofseveral methods which employ induction of in vivo production of antibodymolecules, screening of immunoglobulin libraries (Orlandi. et al, 1989.Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991, Nature349:293-299) or generation of monoclonal antibody molecules by celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the Epstein-Barrvirus (EBV)-hybridoma technique (Kohler et al., 1975. Nature256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote etal., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984.Mol. Cell. Biol. 62:109-120).

Suitable monoclonal antibodies to selected antigens may be prepared byknown techniques, for example those disclosed in “Monoclonal Antibodies:A manual of techniques”, H Zola (CRC Press, 1988) and in “MonoclonalHybridoma Antibodies: Techniques and Applications”, J G R Hurrell (CRCPress, 1982).

Antibody fragments can be obtained using methods well known in the art(see, for example, Harlow & Lane, 1988, “Antibodies: A LaboratoryManual”, Cold Spring Harbor Laboratory, New York). For example, antibodyfragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Alternatively,antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods.

It will be appreciated by persons skilled in the art that for humantherapy or diagnostics, humanised antibodies are preferably used.Humanised forms of non-human (e.g. murine) antibodies are geneticallyengineered chimeric antibodies or antibody fragments having preferablyminimal-portions derived from non-human antibodies. Humanised antibodiesinclude antibodies in which complementary determining regions of a humanantibody (recipient antibody) are replaced by residues from acomplementary determining region of a non-human species (donor antibody)such as mouse, rat of rabbit having the desired functionality. In someinstances, Fv framework residues of the human antibody are replaced bycorresponding non-human residues. Humanised antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported complementarity determining region or framework sequences. Ingeneral, the humanised antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the complementarity determining regions correspondto those of a non-human antibody and all, or substantially all, of theframework regions correspond to those of a relevant human consensussequence. Humanised antibodies optimally also include at least a portionof an antibody constant region, such as an Fc region, typically derivedfrom a human antibody (see, for example, Jones et al., 1986. Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992,Curr. Op. Struct. Biol. 2:593-596).

Methods for humanising non-human antibodies are well known in the art.Generally, the humanised antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues, often referred to as imported residues, aretypically taken from an imported variable domain. Humanisation can beessentially performed as described (see, for example, Jones et al.,1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988, Science 239:1534-15361; U.S. Pat. No. 4,816,567)by substituting human complementarity determining regions withcorresponding rodent complementarity determining regions. Accordingly,such humanised antibodies are chimeric antibodies, wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanisedantibodies may be typically human antibodies in which somecomplementarity determining region residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies.

The CDRs of the antibodies explicitly disclosed herein are of humanorigin. This is advantageous because they are generally less immunogenicthan antibodies of non-human origin.

Human antibodies can also be identified using various techniques knownin the art, including phage display libraries (see, for example,Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J.Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies andCancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol.147:86-95).

In one embodiment there is provided an antibody or antigen-bindingfragment comprising the CDRs, such as 6 CDRs or variable regions from anantibody disclosed herein. The antibody or antigen-binding fragment maybe chimeric. Chimeric antibodies comprise fragments, for exampleframeworks and/or constant regions from a non-human species, for examplemouse, rat, monkey etc. This may be used to prepare a parallel reagent,for example for use in the in vivo safety and/or efficacy studies.

Once suitable antibodies are obtained, they may be tested for activity,for example by ELISA.

In one embodiment of the first aspect of the invention the antibody orantigen-binding fragment, derivative or variant thereof comprises: oneor more CDR sequences selected from SEQ ID NOs: 9-11, 5, 13-14, 36, 30,32, 34, 38, 40, 42, 44, 46, 116 and 118; and/or one or more CDRsequences selected from SEQ ID NOs: 1-3, 5-7, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 135, 88, 90 and114; and/or one or more CDR sequences selected from SEQ ID NOs 16-18, 5,13, 20 and 121; and/or one or more CDR sequences selected from SEQ IDNOs 22-24 and 26-28.

By “CDR” we refer to a complementarity determining region as found in anintact immunoglobulin variable heavy (VH) or variable light (VL) chain.Three complementarity determining regions (CDRs) are present on thevariable domains of both the heavy and light chains of completeimmunoglobulins. The CDRs are numbered as CDR1, CDR2 and CDR3 on boththe heavy and light chains. For example, the VH chain comprises a CDR1,CDR2 and CDR3 and the VL chain also comprises a CDR1, CDR2 and CDR3.

The assignment of amino acids to each CDR region described herein is inaccordance with the definitions according to Kabat E A et al. 1991, In“Sequences of Proteins of Immunological Interest” Fifth Edition, NIHPublication No. 91-3242, pp xv-xvii.

Accordingly, six CDR sequences are most preferably included in theantibody or antigen-binding fragment, derivative or variant thereof.However, fewer CDR sequences, including as few as one, may be included,for example in a single chain antibody fragment.

In a further embodiment the antibody or antigen-binding fragment,derivative or variant thereof comprises: one or more CDR3 sequencesselected from SEQ ID NOs: 11, 14, 36, 30, 32, 34, 38, 40, 42, 44 and 46;and/or one or more CDR3 sequences selected from SEQ ID NOs: 3, 7, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 135, 88 and 90; and/or one or more CDR3 sequences selected from SEQID NOs 18 and 20; and/or one or more CDR3 sequences selected from SEQ IDNOs 24 and 28.

As described above, “CDR3” refers to the third CDR present on either thefull length variable heavy (VH) or full length variable light (VL)antibody chain.

In one embodiment, the antibody or antigen-binding fragment, derivativeor variant thereof comprises: one or more CDR3 sequences selected fromSEQ ID NOs: 11, 36, 30 and 34; and/or one or more CDR3 sequencesselected from SEQ ID NOs: 3, 54, 66 and 70; and/or one or more CDR3sequences selected from SEQ ID NOs: 7, 76, 88 and 90.

In a particular embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH CDR3 sequence selectedfrom SEQ ID NOs: 3, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 and 70; aVH CDR3 sequence selected from SEQ ID NOs: 3, 54, 66 and 70; or a VHCDR3 sequence selected from SEQ ID NOs: 3 and 54.

In another particular embodiment, the antibody or antigen-bindingfragment, derivative or variant thereof comprises: a VL CDR3 sequenceselected from SEQ ID NOs: 7, 72, 74, 76, 78, 80, 82, 84, 86, 135, 88 and90; a VL CDR3 sequence selected from SEQ ID NOs: 7, 76, 88 and 90; or aVL CDR3 sequence of SEQ ID NO 7.

In a further embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises a VH sequence comprising thesequence of SEQ ID NO: 4 or 112, and wherein the VH sequence comprises aCDR3 sequence which is replaced with: a VH CDR3 sequence selected fromSEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 and 70; a VH CDR3sequence selected from SEQ ID NOs: 54, 66 and 70; or a VH CDR3 sequenceof SEQ ID NO: 54.

By “replaced” it is meant that the CDR3 sequence of the V_(H) sequenceis deleted and an alternative CDR3 sequence (as specified) is includedin its place.

In this specific context, by “replaced” it is meant that the CDR3sequence of the VH sequence SEQ ID NO: 4 or 112 is deleted from thesequence and an alternative CDR3 sequence (as specified) is included inits place. In other words, SEQ ID NO: 3 (which is the CDR3 sequence ofSEQ ID NO: 4 and 112) is replaced with an alternative CDR3 sequence (asspecified).

Optionally, the antibody or antigen-binding fragment, derivative orvariant thereof comprises a VL sequence comprising the sequence of SEQID NO: 8, 124, 113 or 139 and wherein the VL sequence comprises a CDR3sequence which is replaced with: a VL CDR3 sequence selected from SEQ IDNOs: 72, 74, 76, 78, 80, 82, 84, 86, 135, 88 and 90; or a VL CDR3sequence selected from SEQ ID NOs: 76, 88 and 90.

By “replaced” in this specific context it is meant that the CDR3sequence of the VL sequence SEQ ID NO: 8, 124, 113 or 139 is deletedfrom the sequence and an alternative CDR3 sequence (as specified) isincluded in its place. In other words, SEQ ID NO: 7 (which is the CDR3sequence of SEQ ID NO: 8, 124, 113 and 139) is replaced with analternative CDR3 sequence (as specified).

In a further embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH CDR3 sequence selectedfrom SEQ ID NOs: 11, 30, 32, 34, 36, 38, 40, 42, 44 and 46; a VH CDR3sequence selected from SEQ ID NOs: 11, 30, 34 and 36; or a VH CDR3sequence selected from SEQ ID NOs 11, 30 and 36.

In one embodiment, the antibody or antigen-binding fragment, derivativeor variant thereof comprises a VL sequence comprising the sequence ofSEQ ID NO: 15, 125, 117 or 140.

In another embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises a VH sequence comprising thesequence of SEQ ID NO: 12 or SEQ ID NO: 115, and wherein the VH sequencecomprises a CDR3 sequence which is replaced with: a VH CDR3 sequenceselected from SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44 and 46; a VHCDR3 sequence selected from SEQ ID NOs: 30, 34 and 36; or a VH CDR3sequence selected from SEQ ID NOs 30 and 36.

By “replaced” in this specific context it is meant that the CDR3sequence of the VH sequence SEQ ID NO: 12 or 115 is deleted from thesequence and an alternative CDR3 sequence (as specified) is included inits place. In other words, SEQ ID NO: 11 (which is the CDR3 sequence ofSEQ ID NO: 12 and 115) is replaced with an alternative CDR3 sequence (asspecified).

In a particular embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VL CDR3 sequence of SEQ IDNO: 14 and a VH CDR3 sequence selected from SEQ ID NOs: 11 and 30-46; orcomprises a VL CDR3 sequence of SEQ ID NO: 7 and a VH CDR3 sequenceselected from SEQ ID NOs: 3 and 48-70; or comprises a VH CDR3 sequenceof SEQ ID NO: 3 and a VL CDR3 sequence selected from SEQ ID NOs: 7 and72, 74, 76, 78, 80, 82, 84, 86, 135, 88 and 90; or comprises a VH CDR3sequence of SEQ ID NO: 18 and a VL CDR3 sequence of SEQ ID NO: 20; orcomprises a VH CDR3 sequence of SEQ ID NO: 24 and a VL CDR3 sequence ofSEQ ID NO: 28.

Preferably, the antibody or antigen-binding fragment, derivative orvariant thereof comprises:

at least one CDR sequence selected from SEQ ID NOs: 1-3, and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 48 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 50 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 52 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 54 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 56 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 58 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 60 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 62 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 64 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 66 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 68 and 5-7; or atleast one CDR sequence selected from SEQ ID NOs: 1, 2, 70 and 5-7; or

at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 72; orat least one CDR sequence selected from SEQ ID NOs: 1-3, 5-6 and 74; orat least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 76; orat least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 78; orat least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 80; orat least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 82; orat least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 84; orat least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 86; orat least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 135;or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 88;or at least one CDR sequence selected from SEQ ID NOs: 1-3, 5, 6 and 90;or at least one CDR selected from SEQ ID NOs: 1-3, 5, 7 and 114; or

at least one CDR sequence selected from SEQ ID NOs: 9-11 and 5, 13 and14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 30, 5,13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10,32, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs:9, 10, 34, 5, 13 and 14; or at least one CDR sequence selected from SEQID NOs: 9, 10, 36, 5, 13 and 14; or at least one CDR sequence selectedfrom SEQ ID NOs: 9, 10, 38, 5, 13 and 14; or at least one CDR sequenceselected from SEQ ID NOs: 9, 10, 40, 5, 13 and 14; or at least one CDRsequence selected from SEQ ID NOs: 9, 10, 42, 5, 13 and 14; or at leastone CDR sequence selected from SEQ ID NOs: 9, 10, 44, 5, 13 and 14; orat least one CDR sequence selected from SEQ ID NOs: 9, 10, 46, 5, 13 and14; or at least one CDR selected from SEQ ID NOs: 9, 11, 116, 5, 14 and118; or

at least one CDR sequence selected from SEQ ID NOs: 16-18 and 5, 13 and20; or at least one CDR sequence selected from SEQ ID NOs: 16-18 and 5,121 and 20; or at least one CDR sequence selected from SEQ ID NOs: 22-24and 26-28.

Each of these groups of sequences correspond to the sequences of the VHCDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3 regions.

The antibody or antigen-binding fragment, derivative or variant thereofmay comprise at least one of the CDR sequences selected from one of thegroups listed above. Preferably, the antibody or antigen-bindingfragment, derivative or variant thereof comprises at least one, two,three, four, five or six CDR sequences selected from one of the groupslisted above. More preferably, the antibody or antigen-binding fragment,derivative or variant thereof comprises at least three or at least sixCDR sequences selected from one of the groups listed above. Mostpreferably, the antibody or antigen-binding fragment, derivative orvariant thereof comprises at least six CDR sequences selected from oneof the groups listed above.

Optionally, the antibody or antigen-binding fragment, derivative orvariant thereof comprises: at least one VH CDR sequence selected fromSEQ ID NOs: 1-3; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 48; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 50; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 52; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 54; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 56; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 58; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 60; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 62; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 64; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 66; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 68; or at least one VH CDR sequence selected from SEQ IDNOs: 1, 2 and 70; or

at least one VH CDR sequence selected from SEQ ID NOs: 9-11; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 30; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 32; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 34; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 36; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 38; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 40; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 42; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 44; or at leastone VH CDR sequence selected from SEQ ID NOs: 9, 10 and 46; or

at least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 11; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 30; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 32; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 34; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 36; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 38; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 40; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 42; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 44; orat least one VH CDR sequence selected from SEQ ID NOs: 9, 116 and 46; or

at least one VH CDR sequence selected from SEQ ID NOs: 16-18; or atleast one VH CDR sequence selected from SEQ ID NOs: 22-24.

Each of these groups of sequences correspond to the sequences of the VHCDR1, CDR2 and CDR3 regions.

The antibody or antigen-binding fragment, derivative or variant thereofmay comprises at least one of the VH CDR sequences selected from one ofthe groups listed above.

Preferably, the antibody or antigen-binding fragment, derivative orvariant thereof comprises at least one, two or three VH CDR sequencesselected from one of the groups listed above.

Most preferably, the antibody or antigen-binding fragment, derivative orvariant thereof comprises at least three VH CDR sequences selected fromone of the groups listed above.

Optionally, the antibody or antigen-binding fragment, derivative orvariant thereof comprises: at least one VL CDR sequence selected fromSEQ ID NOs: 5-7; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 72; or at least one VL CDR sequence selected from SEQ IDNOs: 5-6 and 74; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 76; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 78; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 80; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 82; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 84; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 86; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 135; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 88; or at least one VL CDR sequence selected from SEQ IDNOs: 5, 6 and 90; or

at least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 7; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 72; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 74; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 76; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 78; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 80; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 82; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 84; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 86; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 88; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 114 and 90; or

at least one VL CDR sequence selected from SEQ ID NOs: 5, 13 and 14; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 118 and 14; or

at least one VL CDR sequence selected from SEQ ID NOs: 5, 13 and 20; orat least one VL CDR sequence selected from SEQ ID NOs: 5, 121 and 20; orat least one VL CDR sequence selected from SEQ ID NOs: 26-28.

Each of these groups of sequences correspond to the sequences of the VLCDR1, CDR2 and CDR3 regions.

The antibody or antigen-binding fragment, derivative or variant thereofmay comprises at least one of the VL CDR sequences selected from one ofthe groups listed above. Preferably, the antibody or antigen-bindingfragment, derivative or variant thereof comprises at least one, two orthree VL CDR sequences selected from one of the groups listed above.Most preferably, the antibody or antigen-binding fragment, derivative orvariant thereof comprises at least three VL CDR sequences selected fromone of the groups listed above.

In a particular embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH CDR3 sequence selectedfrom SEQ ID NOs: 3 and 54; or a VH CDR3 sequence selected from SEQ IDNOs: 11, 30 and 36.

In another embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises VH and/or VL sequencescomprising: one or more sequences selected from SEQ ID NOs 12, 15, 125,37, 31, 33, 35, 39, 41, 43, 45, 47, 115, 117 and 140; and/or one or moresequences selected from SEQ ID NOs: 4, 8, 124, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 128, 75, 129, 77, 130, 79, 131, 81, 132, 83,133, 85, 134, 87, 136, 89, 137, 91, 138, 112, 113 and 139; and/or one ormore sequences selected from SEQ ID NOs 19, 21, 126, 119, 120 and 141;and/or one or more sequences selected from SEQ ID NOs 25, 29, 127, 122,123 and 142.

Optionally, the VH sequence of the antibody or antigen-binding fragment,derivative or variant thereof is selected from SEQ ID NOs 12, 37, 31,33, 35, 39, 41, 43, 45, 47 and 115; and/or selected from SEQ ID NOs: 4,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 112; and/or selectedfrom SEQ ID NOs: 19 and 119; and/or selected from: SEQ ID NOs 25 and122.

Optionally, the VL sequence of the antibody or antigen-binding fragment,derivative or variant thereof is selected from SEQ ID NOs: 15, 125, 117and 140; and/or is selected from SEQ ID NOs: 8, 124, 73, 128, 75, 129,77, 130, 79, 131, 81, 132, 83, 133, 85, 134, 87, 136, 89, 137, 91, 138,113 and 139; and/or is selected from SEQ ID NOs: 21, 126, 120 and 141;and/or is selected from SEQ ID NOs: 29, 127, 123 and 142.

In a particular embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises both a VH and a VL sequencecomprising the sequences of a VH and VL sequence pair selected from thesequence pairs: SEQ ID NOs 12, and 15 or 125; SEQ ID NOs 115, and 15 or125; SEQ ID NOs 12, and 117 or 140; SEQ ID NOs 37, and 15 or 125; SEQ IDNOs 37, and 117 or 140; SEQ ID NOs 31, and 15 or 125; SEQ ID NOs 33, and15 or 125; SEQ ID NOs 35, and 15 or 125; SEQ ID NOs 39, and 15 or 125;SEQ ID NOs 41, and 15 or 125; SEQ ID NOs 43, and 15 or 125; SEQ ID NOs45, and 15 or 125; SEQ ID NOs 47, and 15 or 125; SEQ ID NOs 31, and 117or 140; SEQ ID NOs 33, and 117 or 140; SEQ ID NOs 35, and 117 or 140;SEQ ID NOs 39, and 117 or 140; SEQ ID NOs 41, and 117 or 140; SEQ ID NOs43, and 117 or 140; SEQ ID NOs 45, and 117 or 140; SEQ ID NOs 47, and117 or 140; and SEQ ID NOs 115, and 117 or 140; or

selected from the sequence pairs: SEQ ID NOs 4, and 8 or 124; SEQ ID NOs49, and 8 or 124; SEQ ID NOs 51, and 8 or 124; SEQ ID NOs 53, and 8 or124; SEQ ID NOs 55, and 8 or 124; SEQ ID NOs 57, and 8 or 124; SEQ IDNOs 59, and 8 or 124; SEQ ID NOs 61, and 8 or 124; SEQ ID NOs 63, and 8or 124; SEQ ID NOs 65, and 8 or 124; SEQ ID NOs 67, and 8 or 124; SEQ IDNOs 69, and 8 or 124; SEQ ID NOs 71, and 8 or 124; SEQ ID NOs 112, and 8or 124; SEQ ID NOs 4, and 113 or 139; SEQ ID NOs 49, and 113 or 139; SEQID NOs 51, and 113 or 139; SEQ ID NOs 53, and 113 or 139; SEQ ID NOs 55,and 113 or 139; SEQ ID NOs 57, and 113 or 139; SEQ ID NOs 59, and 113 or139; SEQ ID NOs 61, and 113 or 139; SEQ ID NOs 63, and 113 or 139; SEQID NOs 65, and 113 or 139; SEQ ID NOs 67, and 113 or 139; SEQ ID NOs 69,and 113 or 139; SEQ ID NOs 71, and 113 or 139; SEQ ID NOs 4, and 73 or128; SEQ ID NOs 4, and 75 or 129; SEQ ID NOs 4, and 77 or 130; SEQ IDNOs 4, and 79 or 131; SEQ ID NOs 4, and 81 or 132; SEQ ID NOs 4, and 83or 133; SEQ ID NOs 4, and 85 or 134; SEQ ID NOs 4, and 87 or 136; SEQ IDNOs 4, and 89 or 137; SEQ ID NOs 4, and 91 or 138; SEQ ID NOs 112, and73 or 128; SEQ ID NOs 112, and 75 or 129; SEQ ID NOs 112, and 77 or 130;SEQ ID NOs 112, and 79 or 131; SEQ ID NOs 112, and 81 or 132; SEQ ID NOs112, and 83 or 133; SEQ ID NOs 112, and 85 or 134; SEQ ID NOs 112, and87 or 136; SEQ ID NOs 112, and 89 or 137; SEQ ID NOs 112, and 91 or 138;and SEQ ID NOs 112, and 113 or 139; or

selected from the sequence pairs: SEQ ID NOs 19, and 21 or 126; SEQ IDNOs 19, and 120 or 141; SEQ ID NOs 119, and 21 or 126; and SEQ ID NOs119, and 120 or 141; or

selected from the sequence pairs: SEQ ID NOs 25, and 29 or 127; SEQ IDNOs 25, and 123 or 142; SEQ ID NOs 122, and 29 or 127; and SEQ ID NOs122, and 123 or 142.

In other words, the VH and VL sequences of the antibody orantigen-binding fragment, derivative or variant thereof comprise the VHand VL sequences of the specified sequence pairs, i.e. each pair will bemade up of the specified VH sequence and one of the two specified VLsequences.

In a preferred embodiment the antibody or antigen-binding fragment,derivative or variant thereof comprises a VH sequence comprising asequence selected from SEQ ID NOs: 4, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71 and 112.

In another preferred embodiment the antibody or antigen-bindingfragment, derivative or variant thereof comprises a VL sequencecomprising a sequence selected from SEQ ID NOs: 8, 124, 73, 128, 75,129, 77, 130, 79, 131, 81, 132, 83, 133, 85, 134, 87, 136, 89, 137, 91,138, 113 and 139.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 or 48 or 50 or 52 or 54 or 56 or 58 or 60 or 62 or 64 or 66 or68 or 70 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 or 72 or 74 or 76 or 78 or 80 or 82 or 84 or 86 or 88 or 90or 135 for CDRL3. Using this nomenclature, by “CDRH1” we mean the VHchain CDR1, for example.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 48 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 50 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 52 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 54 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 56 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 58 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 60 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 62 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 64 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 66 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 68 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 70 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 7 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 72 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 74 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 76 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 78 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 80 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 82 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 84 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 86 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 88 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 90 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQID NO: 3 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 6 for CDRL2, andSEQ ID NO: 135 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 11 or 30 or 32 or 34 or 38 or 40 or 42 or 44 or 46 for CDRH3, SEQID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2, and SEQ ID NO:

14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 11 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 30 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 11 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 32 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 34 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 38 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 40 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 42 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 44 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 9 for CDRH1, SEQ ID NO: 10 for CDRH2, SEQID NO: 46 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 14 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 16 for CDRH1, SEQ ID NO: 17 for CDRH2, SEQID NO: 18 for CDRH3, SEQ ID NO: 5 for CDRL1, SEQ ID NO: 13 for CDRL2,and SEQ ID NO: 20 for CDRL3.

In one embodiment there is provided an antibody or antigen-bindingfragment, derivative or variant thereof for example with human variableregions comprising SEQ ID NO: 22 for CDRH1, SEQ ID NO: 23 for CDRH2, SEQID NO: 24 for CDRH3, SEQ ID NO: 26 for CDRL1, SEQ ID NO: 27 for CDRL2,and SEQ ID NO: 28 for CDRL3.

In a further preferred embodiment the antibody or antigen-bindingfragment, derivative or variant thereof comprises a VH sequencecomprising the sequence of SEQ ID NO: 4 or 112.

In another preferred embodiment the antibody or antigen-bindingfragment, derivative or variant thereof comprises a VH sequencecomprising the sequence of SEQ ID NO: 55.

In a further preferred embodiment the antibody or antigen-bindingfragment, derivative or variant thereof comprises a VH sequencecomprising a sequence selected from SEQ ID NOs: 12, 31, 33, 35, 37, 39,41, 43, 45, 47 and 115.

In another preferred embodiment the antibody or antigen-bindingfragment, derivative or variant thereof comprises a VH sequencecomprising the sequence of SEQ ID NO: 12 or 115.

In another preferred embodiment the antibody or antigen-bindingfragment, derivative or variant thereof comprises a VH sequencecomprising the sequence of SEQ ID NO: 31.

In another preferred embodiment the antibody or antigen-bindingfragment, derivative or variant thereof comprises a VH sequencecomprising the sequence of SEQ ID NO: 37.

Optionally the antibody or antigen-binding fragment, derivative orvariant thereof additionally comprises a VL sequence comprising thesequence of SEQ ID NO: 8, 124, 113 or 139.

Optionally the antibody or antigen-binding fragment, derivative orvariant thereof additionally comprises a VH sequence comprising thesequence of SEQ ID NO: 4 or 112.

Optionally the antibody or antigen-binding fragment, derivative orvariant thereof additionally comprises a VL sequence comprising thesequence of SEQ ID NO: 15, 125, 117 or 140.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 37; and a VL sequence comprising the sequence ofSEQ ID NO: 15.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 37; and a VL sequence comprising the sequence ofSEQ ID NO: 125.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 31; and a VL sequence comprising the sequence ofSEQ ID NO: 15.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 31; and a VL sequence comprising the sequence ofSEQ ID NO: 125.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 55: and a VL sequence comprising the sequence ofSEQ ID NO: 8.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 55: and a VL sequence comprising the sequence ofSEQ ID NO: 124.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 4: and a VL sequence comprising the sequence ofSEQ ID NO: 8.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 4: and a VL sequence comprising the sequence ofSEQ ID NO: 124.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 12; and a VL sequence comprising the sequence ofSEQ ID NO: 15.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 12; and a VL sequence comprising the sequence ofSEQ ID NO: 125.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 19: and a VL sequence comprising the sequence ofSEQ ID NO: 21.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 19: and a VL sequence comprising the sequence ofSEQ ID NO: 126.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 25; and a VL sequence comprising the sequence ofSEQ ID NO: 29.

In a preferred embodiment, the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VH sequence comprising thesequence of SEQ ID NO: 25; and a VL sequence comprising the sequence ofSEQ ID NO: 127.

In another preferred embodiment, the antibody or antigen-bindingfragment, derivative or variant thereof comprises: a VH CDR1 sequencecorresponding to the sequence of SEQ ID NO: 9; a VH CDR2 sequencecorresponding to the sequence of SEQ ID NO: 10 or SEQ ID NO: 116; a VLCDR1 sequence corresponding to the sequence of SEQ ID NO: 5; a VL CDR2sequence corresponding to the sequence of SEQ ID NO: 13 or SEQ ID NO:118; a VL CDR3 sequence corresponding to the sequence of SEQ ID NO: 14;and a VH CDR3 sequence corresponding to a sequence selected from SEQ IDNOs: 11, 36, 30, 32, 34, 38, 40, 42, 44 or 46. Preferably, the VH CDR3sequence corresponds to the sequence of SEQ ID NO: 11 or SEQ ID NO: 36.Even more preferably, the VH CDR3 sequence corresponds to the sequenceof SEQ ID NO: 36.

In another preferred embodiment, the antibody or antigen-bindingfragment, derivative or variant thereof comprises: a VH CDR1 sequencecorresponding to the sequence of SEQ ID NO: 1; a VH CDR2 sequencecorresponding to the sequence of SEQ ID NO: 2; a VL CDR1 sequencecorresponding to the sequence of SEQ ID NO: 5; a VL CDR2 sequencecorresponding to the sequence of SEQ ID NO: 6 or SEQ ID NO: 114; a VLCDR3 sequence corresponding to the sequence of SEQ ID NO: 7; and a VHCDR3 sequence corresponding to a sequence selected from SEQ ID NOs: 3,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70.

Preferably, the VH CDR3 sequence corresponds to the sequence of SEQ IDNO: 3 or SEQ ID NO: 54. Even more preferably, the VH CDR3 sequencecorresponds to the sequence of SEQ ID NO: 54.

It will be clear to the skilled person that, where the terminal residueis an arginine (R) in the light chain variable region sequencesdisclosed herein, that is the first amino acid of the constant region.Thus in one embodiment there is provided a light chain variable regionsequence wherein the terminal residue (when it is arginine (R) from theconstant region) is absent.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may modulate the biologicalactivity of tenascin-C by altering the transcription, translation and/orbinding properties of tenascin-C.

Such antibodies may be identified using methods well known in the art,such as:

(a) by determining the effect of a test antibody on levels of expressionof tenascin-C, for example by Southern blotting or related hybridisationtechniques;

(b) by determining the effect of a test antibody on levels of tenascin-Cprotein, for example by immunoassays using anti-tenascin-C antibodies;and

(c) by determining the effect of a test antibody on a functional markeror result of tenascin-C activity, for example via the methods of theexamples.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may down-regulate the biologicalactivity of tenascin-C.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may up-regulate the biologicalactivity of tenascin-C. The desirability of up-regulating activity ofimmune and inflammatory molecules and cells is relevant to theproduction of therapies for compromised immune and inflammatory patientsand in the development of vaccines. (see Harandi (2009)).

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may be an inhibitor oftranscription of tenascin-C.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may be an inhibitor of translationof tenascin-C.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may be an inhibitor of the bindingproperties of tenascin-C. For example, the antibody or antigen-bindingfragment, derivative or variant thereof may alter the conformation oftenascin-C such that it is no longer able to bind to its receptor orreceptors.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may be a competitive bindinginhibitor of tenascin-C. It will be appreciated by persons skilled inthe art that the antibody or antigen-binding fragment, derivative orvariant thereof may also inhibit the biological activity of tenascin-Cby blocking tenascin-C receptor function either directly (by acting as atenascin-C receptor antagonist) or indirectly (by binding intermediaryor assisting molecules).

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may be an antagonist of the TLR-4receptor. By an antagonist of TLR4 we include indirect antagonism. Theantigen-binding fragment, derivative or variant thereof might preventtenascin-C activation of TLR4 or also of any other receptor.

It will be appreciated by persons skilled in the art that inhibition ofthe biological activity of tenascin-C by an antibody or antigen-bindingfragment, derivative or variant thereof of the invention may be in wholeor in part. For example, the antibody or antigen-binding fragment,derivative or variant thereof may inhibit the biological activity oftenascin-C by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%,70%, 80% or 90%, and most preferably by 100% compared to the biologicalactivity of tenascin-C on inflammatory cells which have not been exposedto the antibody or antigen-binding fragment, derivative or variantthereof.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may be selected from polyclonal ormonoclonal antibodies.

The antibody or antigen-binding fragment, derivative or variant thereofmay bind substantially reversibly or substantially irreversibly to anactive site of tenascin-C. In a further example, the antibody orantigen-binding fragment, derivative or variant thereof may bind to aportion of tenascin-C that is not the active site so as to interferewith the binding of the tenascin-C to a ligand or receptor. In a stillfurther example, the antibody or antigen-binding fragment, derivative orvariant thereof may bind to a portion of tenascin-C so as to decreasethe proteins activity by an allosteric effect. This allosteric effectmay be an allosteric effect that is involved in the natural regulationof the activity of tenascin-C, for example in the activation of thetenascin-C by an “upstream activator”.

Methods for detecting interactions between a test antibody orantigen-binding fragment, derivative or variant thereof and tenascin-Care well known in the art.

For example ultrafiltration with ion spray mass spectroscopy/HPLCmethods or other physical and analytical methods may be used. Inaddition, Fluorescence Energy Resonance Transfer (FRET) methods may beused, in which binding of two fluorescent labelled entities may bemeasured by measuring the interaction of the fluorescent labels when inclose proximity to each other.

Alternative methods of detecting binding of a polypeptide tomacromolecules, for example DNA, RNA, proteins and phospholipids,include a surface plasmon resonance assay, for example as described inPlant et al., 1995, Analyt Biochem 226(2), 342-348. Methods may make useof a polypeptide that is labelled, for example with a radioactive orfluorescent label.

A further method of identifying an antibody or antigen-binding fragment,derivative or variant thereof that is capable of binding to thepolypeptide is one where the polypeptide is exposed to the antibody orantigen-binding fragment, derivative or variant thereof and any bindingof the compound to the said polypeptide is detected and/or measured. Thebinding constant for the binding of the antibody or antigen-bindingfragment, derivative or variant thereof to the polypeptide may bedetermined. Suitable methods for detecting and/or measuring(quantifying) the binding of an antibody or antigen-binding fragment,derivative or variant thereof to a polypeptide are well known to thoseskilled in the art and may be performed, for example, using a methodcapable of high throughput operation, for example a chip-based method.New technology, called VLSIPS™, has enabled the production of extremelysmall chips that contain hundreds of thousands or more of differentmolecular probes. These biological chips or arrays have probes arrangedin arrays, each probe assigned a specific location. Biological chipshave been produced in which each location has a scale of, for example,ten microns. The chips can be used to determine whether target moleculesinteract with any of the probes on the chip. After exposing the array totarget molecules under selected test conditions, scanning devices canexamine each location in the array and determine whether a targetmolecule has interacted with the probe at that location.

Another method of identifying antibody or antigen-binding fragment,derivative or variant thereof with binding affinity for tenascin-C isthe yeast two-hybrid system, where the polypeptides of the invention canbe used to “capture” proteins that bind tenascin-C. The yeast two-hybridsystem is described in Fields & Song, Nature 340:245-246 (1989).

The antibody or antigen-binding fragment, derivative or variant thereofmay be a high affinity molecule that mimics an antibody (a so-called‘affibody’) (for example, see U.S. Pat. No. 5,831,012 andwww.affibody.se). These ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A (a surface protein from the bacteriumStaphylococcus aureus). This scaffold has excellent features as anaffinity ligand and can be designed to bind with high affinity to anygiven target protein.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may prevent tenascin-C activationof Toll-Like Receptor 4 (TLR4) or other receptors, co-receptors ofToll-Like Receptor 4, or co-receptors of those other receptors.

Co-receptors to primary receptors, such as TLR4, assist with binding ofa signalling molecule to the primary receptor in order to facilitateligand recognition and binding and initiate/maintain the biologicalprocess resulting from receptor binding.

The antibody or antigen-binding fragment, derivative or variant thereofof the first aspect of the invention may preferably have specificity forthe FBG domain of tenascin-C.

In a second aspect of the invention there is provided a compositioncomprising an antibody or antigen-binding fragment, derivative orvariant thereof as defined in the first aspects of the invention and apharmaceutically acceptable carrier, excipient and/or diluent.

It will be appreciated by persons skilled in the art that such aneffective amount of the antibody or antigen-binding fragment, derivativeor variant thereof or formulation thereof may be delivered as a singlebolus dose (i.e. acute administration) or, more preferably, as a seriesof doses over time (i.e. chronic administration).

The antibody or antigen-binding fragment, derivative or variant thereofof the invention can be formulated at various concentrations, dependingon the efficacy/toxicity of the compound being used and the indicationfor which it is being used. Preferably, the formulation comprises theantibody or antigen-binding fragment, derivative or variant thereof ofthe invention at a concentration of between 0.1 μM and 1 mM, morepreferably between 1 μM and 100 μM, between 5 μM and 50 μM, between 10μM and 50 μM, between 20 μM and 40 μM and most preferably about 30 μM.Alternatively, between 60 μM and 70 μM, preferably about 67 μM. For invitro applications, formulations may comprise a lower concentration of acompound of the invention, for example between 0.0025 μM and 1 μM.

It will be appreciated by persons skilled in the art that the antibodyor antigen-binding fragment, derivative or variant thereof of theinvention will generally be administered in admixture with a suitablepharmaceutical excipient diluent or carrier selected with regard to theintended route of administration and standard pharmaceutical practice(for example, see Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company,Pennsylvania, USA).

For example, the antibody or antigen-binding fragment, derivative orvariant thereof of the invention can be administered orally, buccally orsublingually in the form of tablets, capsules, ovules, elixirs,solutions or suspensions, which may contain flavouring or colouringagents, for immediate-, delayed- or controlled-release applications. Theantibody or antigen-binding fragment, derivative or variant thereof ofinvention may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose,lactose, sodium citrate, calcium carbonate, dibasic calcium phosphateand glycine, disintegrants such as starch (preferably corn, potato ortapioca starch), sodium starch glycollate, croscarmellose sodium andcertain complex silicates, and granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia.Additionally, lubricating agents such as magnesium stearate, stearicacid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

The antibody or antigen-binding fragment, derivative or variant thereofof the invention can also be administered parenterally, for example,intravenously, intra-articularly, intra-arterially, intraperitoneally,intrathecally, intraventricularly, intrasternally, intracranially,intra-muscularly or subcutaneously, or they may be administered byinfusion techniques. They are best used in the form of a sterile aqueoussolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. The aqueoussolutions should be suitably buffered (preferably to a pH of from 3 to9), if necessary. The preparation of suitable parenteral formulationsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Example approaches:

-   -   1) Excipients such as buffers and detergents (usually Tween)        that are added to inhibit aggregation in aqueous formulations.    -   2) Freeze drying with appropriate excipients to provide bulk,        stability and cosmetic appeal to the cake    -   3) Formation of a glassy star using compounds such as trehalose.

For oral and parenteral administration, or other routes ofadministration, to human patients, the daily dosage level of theantibody or antigen-binding fragment, derivative or variant thereof ofthe invention will usually be from 1 to 1000 mg per adult (i.e. fromabout 0.015 to 15 mg/kg), administered in single or divided doses.

As an example, the dosage level may be from about 0.5 mg/kg to about 10mg/kg, the administration regimen may be twice or three times weekly,the administration may be intravenous.

The antibody or antigen-binding fragment, derivative or variant thereofof the invention can also be administered intranasally or by inhalationand are conveniently delivered in the form of a dry powder inhaler or anaerosol spray presentation from a pressurised container, pump, spray ornebuliser with the use of a suitable propellant, e.g.dichlorodifluoromethane, trichlorofluoro-methane,dichlorotetrafluoro-ethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas. In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activeantibody or antigen-binding fragment, derivative or variant thereof,e.g. using a mixture of ethanol and the propellant as the solvent, whichmay additionally contain a lubricant, e.g. sorbitan trioleate. Capsulesand cartridges (made, for example, from gelatin) for use in an inhaleror insufflator may be formulated to contain a powder mix of a compoundof the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that eachmetered dose or ‘puff’ contains at least 1 mg of an antibody orantigen-binding fragment, derivative or variant thereof of the inventionfor delivery to the patient. It will be appreciated that the overalldaily dose with an aerosol will vary from patient to patient, and may beadministered in a single dose or, more usually, in divided dosesthroughout the day.

Alternatively, the antibody or antigen-binding fragment, derivative orvariant thereof of the invention can be administered in the form of asuppository or pessary, or they may be applied topically in the form ofa lotion, solution, cream, ointment or dusting powder. The compounds ofthe invention may also be transdermally administered, for example, bythe use of a skin patch. They may also be administered by the ocularroute.

For ophthalmic use, the antibody or antigen-binding fragment, derivativeor variant thereof of the invention can be formulated as micronisedsuspensions in isotonic, pH adjusted, sterile saline, or, preferably, assolutions in isotonic, pH adjusted, sterile saline, optionally incombination with a preservative such as a benzylalkonium chloride.Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the antibody or antigen-bindingfragment, derivative or variant thereof of the invention can beformulated as a suitable ointment containing the active compoundsuspended or dissolved in, for example, a mixture with one or more ofthe following: mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water. Alternatively, they can be formulated as a suitablelotion or cream, suspended or dissolved in, for example, a mixture ofone or more of the following: mineral oil, sorbitan monostearate, apolyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

It may be preferable to use a sustained-release drug delivery system,such as a microspheres. These are designed specifically to reduce thefrequency of injections. An example of such a system is Nutropin Depotwhich encapsulates recombinant human growth hormone (rhGH) inbiodegradable microspheres that, once injected, release rhGH slowly overa sustained period.

Alternatively, the antibody or antigen-binding fragment, derivative orvariant thereof of the present invention can be administered by asurgically implanted device that releases the drug directly to therequired site.

Electroporation therapy (EPT) systems can also be employed for theadministration of the antibody or antigen-binding fragment, derivativeor variant thereof. A device which delivers a pulsed electric field tocells increases the permeability of the cell membranes to the drug,resulting in a significant enhancement of intracellular drug delivery.

The antibody or antigen-binding fragment, derivative or variant thereofcan also be delivered by electroincorporation (EI). EI occurs when smallparticles of up to 30 microns in diameter on the surface of the skinexperience electrical pulses identical or similar to those used inelectroporation. In EI, these particles are driven through the stratumcorneum and into deeper layers of the skin. The particles can be loadedor coated with drugs or genes or can simply act as “bullets” thatgenerate pores in the skin through which the drugs can enter.

An alternative method of antibody or antigen-binding fragment,derivative or variant thereof delivery is the thermo-sensitive ReGelinjectable. Below body temperature, ReGel is an injectable liquid whileat body temperature it immediately forms a gel reservoir that slowlyerodes and dissolves into known, safe, biodegradable polymers. Theactive drug is delivered over time as the biopolymers dissolve.

Antibody or antigen-binding fragment, derivative or variant thereofpharmaceuticals can also be delivered orally. One such system employs anatural process for oral uptake of vitamin B12 in the body to co-deliverproteins and polypeptides. By riding the vitamin B12 uptake system, theprotein or polypeptide can move through the intestinal wall. Complexesare produced between vitamin B12 analogues and the drug that retain bothsignificant affinity for intrinsic factor (IF) in the vitamin B12portion of the complex and significant bioactivity of the drug portionof the complex.

The composition of the second aspect of the invention may furthercomprise at least one other agent.

Such a further agent may be an anti-inflammatory agent which includesbut is not limited to non-steroidal anti-inflammatory agent (NSAID), adisease modifying anti-rheumatic drug (DMARD), a statin (includingHMG-CoA reductase inhibitors such as simvastatin), a biological agent(biologicals), a steroid, an immunosuppressive agent, a salicylateand/or a microbicidal agent. Non-steroidal anti-inflammatory agentsinclude anti-metabolite agents (such as methotrexate) andanti-inflammatory gold agents (including gold sodium thiomalate,aurothiomalate or gold salts, such as auranofin). Biologicals includeanti-TNF agents (including adalimumab, etanercept, infliximab, anti-IL-1reagents, anti-IL-6 reagents, anti-B cell reagents (retoximab), anti-Tcell reagents (anti-CD4 antibodies), anti-IL-15 reagents, anti-CLTA4reagents, anti-RAGE reagents), antibodies, soluble receptors, receptorbinding proteins, cytokine binding proteins, mutant proteins withaltered or attenuated functions, RNAi, polynucleotide aptamers,antisense oligonucleotides or omega 3 fatty acids. Steroids (also knownas corticosteroids) include cortisone, prednisolone or dexamethasone.Immunosuppressive agents include cyclosporin, FK506, rapamycin,mycophenolic acid. Salicylates include aspirin, sodium salicylate,choline salicylate and magnesium salicylate. Microbicidal agents includequinine and chloroquine. For example, the antibody or antigen-bindingfragment, derivative or variant thereof may be administered incombination with one or more of an NSAID, DMARD, or immunosuppressant

In a third aspect of the invention there is provided an antibody orantigen-binding fragment, derivative or variant thereof or compositionas defined in the first and second aspects of the invention for use as amedicament.

In a fourth aspect of the invention there is provided an antibody orantigen-binding fragment, derivative or variant thereof or compositionas defined in the first or second aspects of the invention for use inthe treatment and/or diagnosis of a chronic inflammatory condition.

In a fifth aspect of the invention there is provided the use of anantibody or antigen-binding fragment, derivative or variant thereof orcomposition as defined in as defined in the first or second aspects ofthe invention in the manufacture of a medicament for the treatmentand/or diagnosis of a chronic inflammatory condition.

In a sixth aspect of the invention there is provided a method oftreating a chronic inflammatory condition comprising administering to asubject an effective amount of an antibody or antigen-binding fragment,derivative or variant thereof or composition as defined in the first orsecond aspects of the invention.

The antibody or antigen-binding fragment, derivative or variant thereof,composition, use or method as defined in the third, fourth, fifth orsixth aspects of the invention may relate to treatment of a chronicinflammatory condition wherein the condition is associated with anycondition associated with inappropriate inflammation. Such conditionsinclude, but are not limited to, rheumatoid arthritis (RA), autoimmuneconditions, inflammatory bowel diseases, non-healing wounds, multiplesclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupuserythrematosus (including systemic lupus erythrematosus), asthma,fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UVdamage and psoriasis.

The antibody or antigen-binding fragment, derivative or variant thereofor composition as defined in the first or second aspects may be used,for example, for one or more of the following: to diagnose chronicinflammatory condition status in a subject; to assess the likelihood ofa subject developing a chronic inflammatory condition; to determine theprognosis for a subject with a chronic inflammatory condition; tomonitor disease progression of a chronic inflammatory condition; and/orto monitor effectiveness or response of a subject to a treatment forchronic inflammatory condition.

In a seventh aspect of the invention there is provided an antibody orantigen-binding fragment, derivative or variant thereof or compositionas defined in the first or second aspects for use in the diagnosis of achronic inflammatory condition and/or the determination of prognosis ofa patient with a chronic inflammatory condition.

In an eighth aspect of the invention there is provided a method ofdiagnosing a chronic inflammatory condition and/or determination of theprognosis of a patient with a chronic inflammatory condition comprisingdetecting the presence or absence or amount of the FBG domain oftenascin-C using an antibody or antigen-binding fragment, derivative orvariant thereof or composition as defined in the first or secondaspects.

The prognosis determined may, for example, be a worsening of the chronicinflammatory condition. Alternatively, the prognosis may be a reduction(i.e. improvement) in the chronic inflammatory condition, or theprognosis may be that the chronic inflammatory condition stays the same(i.e. remains constant without worsening or improving).

In one embodiment the method of the eighth aspect is an in vitro method.In an alternative embodiment the method of the eighth aspect is an invivo method.

An increase in the amount of the FBG domain of tenascin-C detected maybe indicative of a chronic inflammatory condition determination and/orof prognosis of a patient with a chronic inflammatory condition.Alternatively, a decrease in the amount of the FBG domain of tenascin-Cdetected may be indicative of a chronic inflammatory conditiondetermination and/or of prognosis of a patient with a chronicinflammatory condition.

Preferably the antibody or antigen-binding fragment, derivative orvariant thereof or composition of the seventh aspect or the method ofthe eighth aspect allows the diagnosis of chronic inflammatory conditionin a subject, or the determination of prognosis of a patient, from theanalysis of the level or amount of the FBG domain of tenascin-C in asample derived from the subject or patient.

In a preferred embodiment of all aspects, the chronic inflammatorycondition is rheumatoid arthritis (RA) and/or erosive rheumatoidarthritis.

The level or amount of FBG domain of tenascin-C present in a samplederived from a subject may be determined by using the antibodies orantigen-binding fragments, derivatives or variants thereof of theinvention in any suitable assay, which may comprise the use of one ormore of: immunoassays; spectrometry; western blot; ELISA;immunoprecipitation; slot or dot blot assay; isoelectric focussing;SDS-PAGE; antibody microarray; immunohistological staining; radio immunoassay (RIA); fluoroimmunoassay; and/or an immunoassay using anavidin-biotin or streptoavidin-biotin system. These methods are wellknown in the art.

By “sample”, we include samples of blood (e.g. serum or plasma),synovial fluid, cerebrospinal fluid (CSF), urine and/or joint tissuederived from the subject.

Preferably, the amount or level of FBG domain of tenascin-C detected iscompared to a reference value in order to determine if the amount orlevel has increased, decreased or stayed the same compared to thatreference value.

Preferably the reference value, to which the detected levels or amountsof the FBG domain of tenascin-C are compared, is the amount or level ofFBG domain of tenascin-C detected in a sample derived from one or moresubjects that do not have any detectable chronic inflammatorycondition/disorder or any clinical symptoms of a chronic inflammatorycondition/disorder (referred to herein as a “normal sample”) and thushave so called “normal values” (also referred to as “normal levels” or“normal amounts”) of the FBG domain of tenascin-C. The actual measuredvalues of those normal levels will depend on the particular assay usedto detect them. However, one example of a normal level/amount (i.e. anormal value) of the FBG domain of tenascin-C present in a sample wouldbe 15-25 ng/ml, preferably 20-21 ng/ml, most preferably 20.7 ng/ml ashas been previously described for tenascin-C levels in Table 2 of Pageet al. (2012), where tenascin C expression in the serum of healthyindividuals and patients with inflammatory conditions has beendescribed.

Preferably an increase of about 50% or more in the level of FBG domainof tenascin-C measured in a sample, compared to the level in a normalsample (i.e. a normal value/level/amount of FBG domain of tenascin-C),is diagnostic of a chronic inflammatory condition or determines theprognosis of a patient with a chronic inflammatory condition. However,in other embodiments, a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or 100% or more increase in the level of FBG domain of tenascin-Cmeasured in a sample, compared to the level in a normal sample, isdiagnostic of a chronic inflammatory condition or determines theprognosis of a patient with a chronic inflammatory condition.

For example, if the level of FBG domain of tenascin-C measured in asample derived from a subject is 50% or more increased from the normallevel of FBG domain of tenascin-C measured in a sample from a healthysubject (i.e. in just one particular example, if it is measured as 31ng/ml or more), the subject is diagnosed as having a chronicinflammatory condition (e.g. RA) and/or the prognosis of that subject isdetermined. The prognosis determined may be a worsening of the chronicinflammatory condition. Alternatively, the prognosis may be a reduction(i.e. improvement) in the chronic inflammatory condition, or theprognosis may be that the chronic inflammatory condition stays the same(i.e. remains constant without worsening or improving).

In a particular embodiment of the antibody or antigen-binding fragment,derivative or variant thereof or method of the seventh and eighthaspects, an increase of at least 50% in the amount of FBG domain oftenascin-C detected compared to normal levels is indicative of a chronicinflammatory condition determination and/or prognosis of a patient witha chronic inflammatory condition.

In a ninth aspect of the invention there is provided an antibody orantigen-binding fragment, derivative or variant thereof or compositionas defined in the first or second aspects for use in the determining theappropriate treatment for an individual, wherein the amount of the FBGdomain of tenascin-C detected indicates the appropriate treatment forthe individual.

In a tenth aspect of the invention there is provided a method ofdetermining the appropriate treatment for an individual comprisingdetecting the presence or absence or amount of the FBG domain oftenascin-C using an antibody or antigen-binding fragment, derivative orvariant thereof or composition as defined in the first or secondaspects, wherein the amount of the FBG domain of tenascin-C detectedindicates the appropriate treatment for the individual.

In one embodiment the method of the tenth aspect is an in vitro method.In an alternative embodiment the method of the tenth aspect is an invivo method.

The appropriate treatment may comprise the administration of aneffective amount of an agent or composition, the agent or compositionmay be one or more of: an antibody or antigen-binding fragment,derivative or variant thereof, or composition as defined in the first orsecond aspects; DMARDS (such as methotrexate); anti-TNF drug; ananti-IL17 therapy; a T-cell co-stimulation modulator (such asOrencia™—abatacept): an interleukin-6 (IL-6) inhibitor (such asActemra™—tocilizumab); an anti-CD20 antibody (such asRituxan™—rituxumab; a B cell activating factor (such as anti-BAFF); aninhibitor of janus kinase (JAK) (such as Tofacitinib™); an inhibitor ofspleen tyrosine kinase (Syk) (such as Fostamatinib™); antiTNC antibodiesor antibodies to citrullinated tenascin-C domains; and/or an agent thatmodulates the biological activity of citrullinated and/ornon-citrullinated tenascin-C.

In a particular embodiment, the appropriate treatment targets the FBGdomain of tenascin-C.

In another particular embodiment, the appropriate treatment is theadministration of an effective amount of an antibody or antigen-bindingfragment, derivative or variant thereof, or composition as defined inthe first or second aspects.

Optionally, the individual defined in the ninth and tenth aspects has achronic inflammatory condition. The individual may or may not have beendiagnosed as such prior to the method being performed.

In certain embodiments, an increase in the amount of FBG domain oftenascin-C detected indicates the appropriate treatment. In alternativeembodiments, a decrease in the amount of FBG domain of tenascin-Cdetected indicates the appropriate treatment.

In one embodiment an increase or decrease of about 50% or more in thelevel of FBG domain of tenascin-C measured in a sample, compared to thelevel in a normal sample (i.e. a normal level or amount of FBG domain oftenascin-C), determines the appropriate treatment of an individual.However, in other embodiments, a 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 100% or more increase or decrease in the level of FBG domainof tenascin-C measured in a sample, compared to the level in a normalsample, determines the appropriate treatment of an individual.

For example, if the level of FBG domain of tenascin-C measured in asample derived from a subject is 50% or greater increased from thenormal level of FBG domain of tenascin-C measured in a sample from ahealthy subject (i.e. in just one particular example, if it is measuredas 31 ng/ml or more), the appropriate treatment is determined. Forexample, it may then be determined to treat the subject by theadministration of an effective amount of an agent or composition, theagent or composition may be one or more of: an antibody orantigen-binding fragment, derivative or variant thereof, or compositionas defined in the first or second aspects; DMARDS (such asmethotrexate); anti-TNF drug; an anti-IL17 therapy; a T-cellco-stimulation modulator (such as Orencia™—abatacept): an interleukin-6(IL-6) inhibitor (such as Actemra™—tocilizumab); an anti-CD20 antibody(such as Rituxan™—rituxumab; a B cell activating factor (such asanti-BAFF); an inhibitor of janus kinase (JAK) (such as Tofacitinib™);an inhibitor of spleen tyrosine kinase (Syk) (such as Fostamatinib™);antiTNC antibodies or antibodies to citrullinated tenascin-C domains,and/or an agent that modulates the biological activity of citrullinatedand/or non-citrullinated tenascin-C.

In one embodiment, an increase in FBG domain of tenascin-C detectedindicates that an increased amount of the appropriate treatment isrequired. In an alternative embodiment, a decrease in FBG domain oftenascin-C detected indicates that an increased amount of theappropriate treatment is required.

Preferably an increase or decrease of 50% or more in the level of FBGdomain of tenascin-C measured in a sample, compared to the level in anormal sample (i.e. a normal level or amount of FBG domain oftenascin-C), indicates that an increased or decreased amount of theappropriate treatment is required. However, in other embodiments, a 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more increase ordecrease in the level of FBG domain of tenascin-C measured in a sample,compared to the level in a normal sample, indicates that an increased ordecreased amount of the appropriate treatment is required. The increasedor decreased amount of the appropriate treatment may be an increase ordecrease in dose, frequency of dosing or duration of treatment.

In one embodiment of the antibody or antigen-binding fragment,derivative or variant thereof or composition or method of ninth andtenth aspects, an increase of at least 50% in the amount of FBG domainof tenascin-C detected compared to normal levels of FBG domain oftenascin-C determines the appropriate treatment and/or indicates that anincreased amount of the appropriate treatment is required.

Conveniently, the method of diagnosis or method of determining theappropriate treatment of the eighth and/or tenth aspects comprisesperforming one or more of: immunoassays; spectrometry; western blot;ELISA; immunoprecipitation; slot or dot blot assay; isoelectricfocussing; SDS-PAGE; antibody microarray; immunohistological staining;radio immuno assay (RIA); fluoroimmunoassay; and/or an immunoassay usingan avidin-biotin or streptoavidin-biotin system.

The antibody or antigen-binding fragment, derivative or variant thereof,composition or method as defined in the seventh, eighth, ninth or tenthaspects of the invention may relate to treatment of a chronicinflammatory condition wherein the condition is associated with anycondition associated with inappropriate inflammation. Such conditionsinclude, but are not limited to, rheumatoid arthritis (RA), autoimmuneconditions, inflammatory bowel diseases, non-healing wounds, multiplesclerosis, cancer, atherosclerosis, sjogrens disease, diabetes, lupuserythrematosus (including systemic lupus erythrematosus), asthma,fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UVdamage and psoriasis.

In an eleventh aspect of the invention there is provided a kit of partscomprising:

-   -   (i) an antibody or antigen-binding fragment, derivative or        variant thereof or composition as defined in the first or second        aspects of the invention    -   (ii) administration means    -   (iii) instructions for their use    -   The kit of the seventh aspect of the invention may further        optionally comprise    -   (iv) at least one other agent.

According to a further aspect of the invention there is provided a kitof parts for use in determining the chronic inflammatory conditionstatus of a subject comprising:

-   -   (i) an antibody or antigen-binding fragment, derivative or        variant thereof or composition as defined in the first or second        aspects of the invention; and    -   (ii) instructions for use

By “chronic inflammatory condition status”, we include the diagnosis of,determining the prognosis of and/or determining the appropriatetreatment for a subject with or without a chronic inflammatorycondition.

Further aspects of the invention relate to methods of identifying one ormore therapeutic antibodies with specificity for FBG, such as theantibodies described in the earlier aspects (see the detailed antibodyscreening methodology described in the Examples).

A detailed specification for the final therapeutic molecule is importantprior to initiating an antibody isolation project. For the antibodiesisolated herein, the set specifications are provided in the table below:

TABLE A Specifications for antibody selection. First Milestones SecondMilestones* (Lead Isolation) (lead optimisation) Panel At least 2antibody leads with unique 1 optimised preclinical development CDRsequence combinations candidate and 1 backup with different CDR sequenceFormat Fab or whole antibody (human IgG2 Human IgG2 or IgG4 or humanIgG4) Affinity Binding affinity of 1-10 nM, Either: Binding affinity of≤320 pM, Potency determined by Surface plasmon determined by SPR; or:Resonance (SPR) IC50 1 nM (at least n = 3) or lower in Displaysconcentration-related assay of tenascin-C - evoked cytokine inhibitionof tenascin-C - evoked release in a cell-based assay cytokine releasewith IC50 < 100 nM in a cell-based assay Cross In ELISA, binds to human,mouse, Affinity to at least one rodent (e.g. rat) Reactivity rat, dogFBG and one non-rodent (e.g. dog) tenascin-C isoform of ≤3 nMSpecificity Evidence of non- binding to human Concentration forhalf-maximal tenascin-R, vs human tenascin-C binding to human tenascin-Ris at positive control (provided antigen is least 50-fold, preferably100-fold, available at the time of reaching greater than equivalentbinding signal other specifications), preferably in to human tenascin-C,preferably in ELISA, or by SPR. ELISA, or by SPR. Solubility Soluble toat least 1 mg/mL in PBS IgG is soluble in PBS to at least 20 mg/mLwithout precipitation/ aggregation over 14 days *or if there isagreement that an acceptable profile is achieved before the “SecondMilestones” criteria are met

The key requirements were identification of antibodies with highaffinity for Tenascin C FBG, able to block cell activation andproduction of inflammatory cytokines in response to Tenascin C FBG, andwith sufficient cross reactivity to other species to allow relevantsafety and efficacy studies to be conducted in those species (and henceno requirements for parallel reagents).

It was also deemed advantageous to have lead antibodies that had a loweraffinity for other members of the Tenascin family and Tenascin R wastherefore chosen for specificity testing based on this protein having ahigher degree of homology (at the amino acid level) to Tenascin C thanother closely related proteins. Cellular potency was defined as the halfmaximal concentration able to inhibit the Tenascin C FBG derivedcytokine release in a set of relevant cell based assays. Finally, thecandidates needed to be stable in relevant antibody formats to providesome early indications that there were no critical issues in themanufacturability of the product.

The screening methods adopted were based on generating antibodies withthe correct specification. Therefore initial ELISA screeningincorporated testing of binding human, rat, mouse and dog TNC-FBG.Positive clones were also tested against human Tenascin R FBG. Thosewith the correct binding properties were subcloned into suitableantibody formats for testing in the potency screen (inhibition of theFc-His-FBG induced alkaline phosphatase reporter in the THP-1 Blue™assay was utilized for screening and then activity was confirmed bymeasuring the inhibition of Fc-His-FBG induced cytokine expression fromTHP-1 cells). Lead clones that passed the first milestone were takeninto lead optimization where the phage display methods were tailored forprovide antibodies that passed the 2^(nd) milestone. Of the ‘parent’antibodies described herein that were taken into lead optimization (2A5,B12, F3 and D8) two (2A5 and B12) provided optimized clones that passedthe pre-agreed specification.

Therefore, in a further aspect of the invention there is provided amethod of identifying one or more therapeutic antibodies specific forthe FBG domain of tenascin-C comprising selecting antibodies which haveone or more of the properties listed in Table A. Preferably theantibodies exhibit all of the properties listed under “FirstMilestones”, even more preferably the antibodies exhibit all of theproperties in Table A.

In one embodiment, the method of of identifying one or more therapeuticantibodies specific for the FBG domain of tenascin-C comprises selectingantibodies which have one or more of the following properties:

-   -   (a) the antibody is a Fab or whole antibody (preferably human        IgG2 or human IgG4);    -   (b) the antibody has a binding affinity to human tenascin-C FBG        of 1-10 nM, determined by Surface plasmon Resonance (SPR),        and/or the antibody displays concentration-related inhibition of        tenascin-C, preferably evoked cytokine release with IC50<100 nM        in a cell-based assay, preferably using Fc-His-FBG; and    -   (c) the antibody binds to human FBG and one or more of mouse,        rat and dog FBG, preferably measured by ELISA, preferably the        antibody binds to all of human, mouse, rat and dog FBG.

Preferably, the antibody also exhibits one or more of the followingadditional properties:

-   -   (d) the antibody does not bind to human tenascin-R, or exhibits        reduced binding to human tenascin-R compared to human tenascin-C        positive control; and    -   (e) The antibody is soluble to at least 1 mg/mL in PBS.

Preferably, the affinity potency determined by SPR is ≤320 pM and/orthere is an IC50 of ≤1 nM for evoked cytokine release in a cell basedassay, preferably using Fc-His-FBG.

Preferably, the antibody has affinity to at least one rodent (e.g. rat)and one non-rodent (e.g. dog) tenascin-C isoform of 3 nM.

Preferably, concentration for half-maximal binding to human tenascin-Ris at least 50-fold, preferably 100-fold, greater than equivalentbinding signal to human tenascin-C, preferably measured by ELISA bindingexperiments, or by SPR. Preferably, the concentration for half-maximalbinding to human tenascin-R is at least 50-fold greater than equivalentbinding signal to human tenascin-C when measured by SPR. Preferably, theconcentration for half-maximal binding to human tenascin-R is at least100-fold greater than equivalent binding signal to human tenascin-C whenmeasured by ELISA.

Preferably, the antibody is soluble in PBS to at least 20 mg/mL withoutprecipitation/aggregation over 14 days.

In a further aspect of the invention there is provided a method ofidentifying one or more therapeutic antibodies specific for the FBGdomain of tenascin-C comprising the following steps:

-   -   (i) Screening an antibody or antibody fragment library, e.g. a        phage library, for antibodies or fragments which bind human        tenascin-C FBG and one or more of rat, mouse and dog tenascin-C        FBG, preferably by ELISA;    -   (ii) Testing positive antibodies or fragments identified in (i)        for reduced binding to human tenascin-R FBG compared to human        tenascin-C FBG, preferably by ELISA;    -   (iii) Subcloning antibodies or fragments identified with the        desired properties from (i) and (ii) into suitable formats for        potency screen (e.g. Fab, Fc-scFv, IgG2 or IgG4); and    -   (iv) Identifying antibodies or fragments from (iii) which        exhibit inhibition of Fc-His-FBG activity.

Optionally, Surface plasmon Resonance (SPR) is used for step (i) and/or(ii).

Preferably step (iv) utilizes an Fc-His-FBG induced alkaline phosphatasereporter (e.g. in the THP-1 Blue™ assay); and/or measures the inhibitionof Fc-His-FBG induced cytokine expression (e.g. from THP-1 cells).

In step (i), preferably the antibody or fragment binds to all of human,rat, mouse and dog tenascin-C FBG.

Optionally the method steps are performed in order, however they mayalternatively be performed in any order.

In one embodiment, the therapeutic antibody or antibodies identified byperforming the method of steps (i)-(iv) exhibits one or more of theproperties (a)-(e) of the previous aspect.

Definitions

By “inflammation” we include the meaning of local accumulation of fluid,plasma proteins, and white blood cells that is initiated by tissueinjury, infection or a local immune response.

By “acute inflammation” we include the meaning of the initial stages(initiation) of inflammation and the short-term transient inflammatoryresponse immediately after injury, infection or local immune response.Typically, acute inflammation is rapidly resolved, lasting from a matterof minutes to no longer that a few days.

By “chronic inflammation” we include the meaning of persistent and/ornon-resolved inflammation. It is often associated with inappropriatedestruction of healthy tissue. This may be progressive and last over aperiod of weeks or longer. Chronic inflammation is typically associatedwith persistent infection or disease including, but not limited to,autoimmune conditions.

By “chronic joint inflammation” we include the meaning of persistentinflammation that is progressive and unremitting over a period of weeksto months, resulting in distortion of the affected joint andradiographic evidence of cartilage and bone destruction as observed inhuman disease (Kelly, Harris, Ruddy and Sledge, Textbook of Rheumatology4th Edition).

In experimental murine models, chronic joint inflammation ischaracterised by inflammation that does not subside and causesinappropriate tissue destruction, even over a relatively short period oftime. This is characterised (and can be identified) histologically bythe prolonged presence of inflammatory cells in the synovium and jointspace, chondrocyte death, and cartilage and bone erosion.

By “fragment” we mean at least four amino acids, for example at least 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50 amino acids.

The percent sequence identity between two polypeptides may be determinedusing suitable computer programs, for example the GAP program of theUniversity of Wisconsin Genetic Computing Group and it will beappreciated that percent identity is calculated in relation topolynucleotides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal Wprogram (as described in Thompson et al., 1994, Nuc. Acid Res.22:4673-4680).

The parameters used may be as follows:

Fast pairwise alignment parameters: K-tuple (word) size; 1, window size;5, gap penalty; 3, number of top diagonals; 5. Scoring method: xpercent.

Multiple alignment parameters: gap open penalty; 10, gap extensionpenalty; 0.05.

Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine localsequence alignments.

By “antibody” we include substantially intact antibody molecules, aswell as chimeric antibodies, humanised antibodies, human antibodies(wherein at least one amino acid is mutated relative to the naturallyoccurring human antibodies), single chain antibodies, bispecificantibodies, antibody heavy chains, antibody light chains, homodimers andheterodimers of antibody heavy and/or light chains, and antigen bindingfragments and derivatives of the same.

By “antigen-binding fragment” we mean a functional fragment of anantibody that is capable of binding to the FBG domain of tenascin-C.

The term “subject” or “individual” means all animals including humans.Examples of subjects include humans, cows, dogs, cats, goats, sheep, andpigs. The term “patient” means a subject or individual having a disorderin need of treatment.

As used herein, ‘pharmaceutical formulation’ means a therapeuticallyeffective formulation according to the invention.

A ‘therapeutically effective amount’, or ‘effective amount’, or‘therapeutically effective’, as used herein, refers to that amount whichprovides a therapeutic effect for a given condition and administrationregimen. This is a predetermined quantity of active material calculatedto produce a desired therapeutic effect in association with the requiredadditive and diluent, i.e. a carrier or administration vehicle. Further,it is intended to mean an amount sufficient to reduce and mostpreferably prevent, a clinically significant deficit in the activity,function and response of the host. Alternatively, a therapeuticallyeffective amount is sufficient to cause an improvement in a clinicallysignificant condition in a host. As is appreciated by those skilled inthe art, the amount of a compound may vary depending on its specificactivity. Suitable dosage amounts may contain a predetermined quantityof active composition calculated to produce the desired therapeuticeffect in association with the required diluent. In the methods and usefor manufacture of compositions of the invention, a therapeuticallyeffective amount of the active component is provided. A therapeuticallyeffective amount can be determined by the ordinary skilled medical orveterinary worker based on patient characteristics, such as age, weight,sex, condition, complications, other diseases, etc., as is well known inthe art.

Examples embodying an aspect of the invention will now be described withreference to the following figures:

FIG. 1. Amino acid sequence of human tenascin-C and its domains: TAdomain (SEQ ID NO: 144), EGFL domain (SEQ ID NO: 145), FNIII domain (SEQID NO: 146), FBG domain (SEQ ID NO: 92).

FIG. 2. Nucleotide sequence of human tenascin-C(SEQ ID NO: 148).

FIG. 3. Polyclonal phage ELISA.

Polyclonal derived 2nd round output phage were incubated with wellscoated with antigen or fusion partner (Fc or Cd4) and bound phagedetected with anti-M13 mAb and Europium-labelled anti-mouse antibody.There is enrichment of antigen-specific binders between rounds 1 and 2of selection and a greater proportion of huFBG binders compared toanti-Fc or-rCd4 phage in the round 2 output populations.

FIGS. 4A-4B. Screening of purified anti-FBG in the THP1-Blue assay ofsecreted alkaline phosphatase release, determined by fluorimetric assay.

Antibodies were tested at the highest concentration achievable. In aconfirmatory assay of purified scFv-Fc clones, 2A3, 2A5, 2611 and 2D12were identified as effective blockers of signalling evoked by 10 nMFc-His-huFBG (FIG. 4A). Assay of purified anti-FBG FAbs highlighted anumber of additional hits for further analysis including antibodies A12,B12, C2, D7, D8, F3 and G1. In this experiment cells were stimulatedwith 3 nM Fc-His-huFBG (FIG. 4B).

FIG. 5. Cross-reactivity ELISA results for purified Fab binding toimmobilised TNC FBG-rCD4 proteins (human, mouse, rat and dog) and humanTNR FBG-rCD4.

Binding was detected using anti-kappa or anti-lambda mAb followed byEuropium-conjugated anti-mouse mAb.

FIGS. 6A-6D illustrate BiAcore sensogram traces for determination ofkinetics for binding of FBG proteins to anti-FBG Fabs B12 (FIG. 6A), 2A5(FIG. 6B), F3 (FIG. 6C), and D8 (FIG. 6D) which were captured on a CM5sensor chip.

Traces indicate binding of human, rat and mouse tenascin-C rCd4-FBG andhuman tenascin-R rCD4-FBG.

FIG. 7. Concentration-related inhibition of secreted alkalinephosphatase (SEAP) release by anti-FBG Fabs B12, 2A5, D8 and F3.

Purified antibodies were incubated with THP1-Blue cells in the presenceof 3 nM human Fc-His-FBG for 18 h at 37° C. IC50 values for inhibitionof Fc-His-FBG evoked SEAP were B12 (1.7 nM), 2A5 (20.6 nM), D8 (7.2 nM),F3 (8.4 nM).

FIG. 8. Concentration-related inhibition of IL-8 production by anti-FBGFabs B12, 2A5, D8 and F3.

Purified antibodies were incubated with THP1-Blue cells in the presenceof 3 nM human Fc-His-FBG for 18 h at 37° C. IC50 values for inhibitionof Fc-His-FBG evoked IL-8 release were B12 (6.9 nM), 2A5 (28.5 nM), D8(14.9 nM), F3 (13.8 nM).

FIGS. 9A-9B. CDR3 randomisation strategy for anti-FBG lead antibodies.FIG. 9A: F3VLCDR3 (SEQ ID NO: 28), 2A5VHCDR3 (SEQ ID NO: 3), 2A5VLCDR3(SEQ ID NO: 7), B12VHCDR3 (SEQ ID NO: 11), B12VLCDR3 (SEQ ID NO: 14),F3VHCDR3 (SEQ ID NO: 24) and oligonucleotides for VH and VL CDR3mutagenesis (SEQ ID NOs: 97-111).

VH CDR3 randomisation was done in three overlapping blocks of 6 residueseach (labelled VH 3.1, VH 3.2, and VH 3.3) and the VL CDR3s wererandomised in blocks of two (labelled VL 3.1 and VL 3.2). Arrowsindicate the positions of stop codons introduced into the template DNAto eliminate parental clones dominating the library. FIG. 9B:Oligonucleotides used for CDR3 library generation.

FIGS. 10A-10B. Selection strategy for CDR randomised antibody libraries.

(FIG. 10A) Selections on human rCd4-His-FBG using CDR randomisedlibraries. (FIG. 10B) Hybrid selections on human and mouse FBG using B12VH and VL CDR 3 randomised libraries.

FIG. 11. Schematic diagram of the anti-FLAG capture ELISA format used toscreen affinity-matured clones for improved binding to biotin-labelledmouse or human rCd4-FBG.

FIGS. 12A-12B. Inhibition of FBG-evoked cytokine release by theaffinity-matured hIgG4 antibodies 165_13_131, 165_13_C3, and 160_01_A4.

Primary human PBMCs were incubated (37° C., 24 h) in the presence of 200nM human Fc-His-FBG and test antibody (100 nM or 1 μM) and supernatantswere assayed for IL-8 (FIG. 12A) and TNFα (FIG. 12B). All testantibodies blocked evoked cytokine release. Data indicate mean±s.e. meanof results from 3 separate donors.

FIGS. 13A-13D. Immunostaining of endogenous tenascin-c FBG in fixedfrozen sections of knee synovium from a rheumatoid arthritis patientfollowing knee replacement surgery.

Specific staining of the synovium was seen with positive controlanti-tenascin-antibody (FIG. 13A), and B12 anti-FBG formatted as mouseIgG2a (FIG. 13C). Lower levels of non-specific staining were observedwith non-immune isotype control antibodies (FIG. 13B, FIG. 13D).

FIGS. 14A-14D. Antibodies C3 (165_13_C3) and B12 show good specificityfor TNC-FBG when used for western blot analysis.

Recombinant TNC-FBG (Nascient), TNR-FBG or FIBRINOGEN (Kennedy Instituteof Rheumatology (KIR)) detected with the following antibodies FIG. 14A)165_13_C3 IgG4 MAb at 1:20,000 (0.25 ug/ml), overnight at 4° C. FIG.14B) B12 IgG4 MAb at 1:20,000 (0.25 ug/ml), overnight at 4° C. FIG. 14C)Anti-Tenascin-R antibody (Santa Cruz Biotechnology, sc-9875) at 1:2,000(0.1 ug/ml) overnight at 4° C. FIG. 14D) Anti-TNC-FBG polyclonalantibody (Midwood group) at 1:500, overnight at 4° C.

FIGS. 15A-15B. Western blot analysis of glioma cell lysate usingmonoclonal antibody B12 and corresponding isotype control.

Glioma cell lysate (KIR) and tenascin-C(Nascient) detected with FIG.15A. B12 IgG4 Mab at 1:20,000, overnight at 4° C.; FIG. 15B. IgG4isotype control (Eureka therapeutics) at 1:4,000, overnight at 4° C.

FIG. 16A. Fc-His-FBG binds to TLR4 in vitro in a dose dependent manner.Recombinant human TLR4 (R&D systems) in PBS (or PBS alone) was bound toa 96-well plate, after blocking the indicated concentrations of HumanFc-His-FBG. was added and detection was carried out by incubation of ananti-human IgG1 MAb (AbD Serotec, clone 2C11) at 1 ug/ml, an anti-mouseHRP conjugated secondary antibody (AbD Serotec, STAR13B) at 1 ug/ml, andTMB substrate. n=4 mean and SEM shown.

FIG. 16B. Monoclonal Ab C3 (165_13_C3) disrupts the binding FBG and TLR4in vitro. Recombinant human TLR4 in PBS (or PBS alone) was bound to a96-well plate, after blocking recombinant human Fc-His-TNC-FBG (100 nM)which had been pre-incubated with C3 Mab or isotype control antibody wasadded. Detection was carried out by successive incubation of antibodydirected against the Fc portion of the protein, an anti-mouse HRPconjugated secondary antibody and TMB substrate. The percentageinhibition in the C3 pre-incubated samples was calculated compared tothe isotype control samples (IC50=44.5 nM). n=4

FIGS. 17A-17C. Monoclonal antibody C3 (165_13_C3) reduces the productionof pro-inflammatory cytokines by primary human macrophages stimulatedwith human or mouse TNC-FBG, but not LPS.

(FIG. 17A) Recombinant Human tenascin-C FBG (1 uM) or LPS (Enzo) (1ng/ml) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2, and 0.04uM) or isotype control MAb (1 uM) before being added in triplicate toHuman M2 macrophage cultures. After 24 h supernatants were taken andsubjected to IL-8, IL-6 and TNF cytokine ELISA (BD Biosciences). n=3;(FIG. 17B) Recombinant Murine tenascin-C FBG (1 uM) was pre-incubatedfor 30 min at RT with MAb C3 (1, 0.2 and 0.04 uM) or isotype control MAb(1 uM) before being added in triplicate to Human M2 macrophage cultures.After 24 h supernatants were taken and subjected to cytokine ELISA. n=3or over, mean and SEM shown; (FIG. 17C) A protein where the Fc portionis mutated to be inactive (Fc-Mut-His-FBG) was used. Other promisinganti-TNC-FBG antibodies, B12 and A4 were also tested in this system.Fc-Mut-His-FBG (1 uM) and C3, 160_01_A4 or B12 (1 uM) were pre-incubatedfor 30 min at RT before being added to human M2 macrophage cultures.After 24 h supernatants were taken and subjected to cytokine ELISA. n=3,mean and SEM shown.

FIG. 18A. Monoclonal antibody B12 reduces the production ofpro-inflammatory cytokines by primary human macrophages stimulated withhuman TNC-FBG.

Recombinant Human tenascin-C FBG (1 uM) was pre-incubated with MAb B12(1, 0.1, 0.01 or 0.001 uM) or isotype control MAb (1 uM) before beingadded in triplicate to Human M2 macrophage cultures. After 24 hsupernatants were taken and subjected to cytokine ELISA.

FIG. 18B. Monoclonal antibody C3 (165_13_C3) produced at laboratory andlarger scale show the same level of efficacy in blockade of FBG-inducedcytokine synthesis by primary human macrophages.

The potency of the antibody produced at larger scale was compared tothat produced by at laboratory scale. Recombinant Human tenascin-C FBG(1 uM) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2 and 0.04uM) or isotype control MAb (1 uM) before being added in triplicate toHuman M2 macrophage cultures. After 24 h supernatants were taken andsubjected to cytokine ELISA. n=1, Ico=laboratory scale, Lon=largerscale.

FIG. 19. Monoclonal antibody C3 (165_13_C3) reduces the production ofpro-inflammatory cytokines by RA synovial fibroblasts stimulated withhuman TNC-FBG.

Recombinant Human tenascin-C FBG (1 uM) was pre-incubated for 30 min atRT with MAb C3 or isotype control MAb before being added in triplicateto Human synovial fibroblast cultures from RA patients. After 24 hsupernatants were taken and subjected to cytokine ELISA. n=1, mean andSEM shown

FIG. 20. Levels of Tenascin-C in rat CIA model.

TNC levels in synovial fluid from a rat model of collagen inducedarthritis. The amount of TNC measured is shown plotted against thecorresponding clinical score for each paw.

FIG. 21. Clinical scores from evaluation of C3 (165_13_C3) antibody in arat model of collagen-induced arthritis.

Vehicle versus 1 mg/kg, 3 mg/kg and 10 mg/kg C3 antibody. Data arepresented as Mean±SEM. Statistical significances: #### p<0.0001 whencompared to Day 7, ** p<0.01 when compared to the vehicle-treated group.

FIG. 22: Hind paw volumes from evaluation of C3 (165_13_C3) antibody ina rat model of collagen-induced arthritis.

Vehicle versus 1 mg/kg, 3 mg/kg and 10 mg/kg C3 antibody. Data arepresented as Mean±SEM. Statistical significances: ## p<0.01 and ####p<0.0001 when compared to Day 0, * p<0.05 and ** p<0.01 when compared tothe vehicle-treated group.

FIG. 23. Primers used for antigen cloning: human FGB-X forward primerminus 2561 (SEQ ID NO: 149), human FBG-X forward primer plus 2565 (SEQID NO: 150), mouse FBG-X forward primer minus 2561 (SEQ ID NO: 151),mouse FBG-X forward primer plus 2566 (SEQ ID NO: 152), human X-FBGforward primer minus 2567C (SEQ ID NO: 153), human X-FBG forward primerplus 2567 (SEQ ID NO: 154), mouse X-FBG forward primer minus 2569C (SEQID NO: 155), mouse X-FBG forward primer primer plus 2568C (SEQ ID NO:156), BamHI-His6-HindIII forward primer 2574 (SEQ ID NO: 157), humanHis-FBG forward primer plus 2580 (SEQ ID NO: 158), mouse His-FBG forwardprimer plus 2580 (SEQ ID NO: 159), human FBG-X reverse primer minus 2562(SEQ ID NO: 160), human FBG-X reverse primer plus 2562 (SEQ ID NO: 161),mouse FBG-X reverse primer minus 2562 (SEQ ID NO: 162), mouse FBG-Xreverse primer plus 2562 (SEQ ID NO: 163), human X-FBG reverse primerminus 2570 (SEQ ID NO: 164), human X-FBG reverse primer plus 2570 (SEQID NO: 165), mouse X-FBG reverse primer minus 2571 (SEQ ID NO: 166),mouse X-FBG reverse primer plus 2571 (SEQ ID NO: 167),BamHI-His6-HindIII reverse primer 2575 (SEQ ID NO: 168), human His-FBGreverse primer plus 2570 (SEQ ID NO: 169), mouse His-FBG reverse primerplus 2571 (SEQ ID NO: 170).

This table details the primers used to generate expression constructsfor use in antigen cloning.

Example 1—Generation of Purified Tenascin-c FBG as Antigen and AssayReagents

Purified soluble proteins containing the FBG domain of tenascin-C (TNCFBG) were generated for use as antigens in antibody selections and asreagents in subsequent screening and characterisation assays. To enableselection strategies for isolation of antibodies that bind tenascin-C ofmultiple mammalian species, a range of DNA expression constructs weresynthesised, which incorporated the TNC FBG domain of either human [SEQID NO: 92], mouse [SEQ ID NO: 93], rat [SEQ ID NO: 94] or dog [SEQ IDNO: 95]. A human tenascin-R FBG [SEQ ID NO: 96] construct was alsoprepared for identification of antibodies that displayed unwantedbinding to this homologue. Constructs were produced as 6His-taggedproteins with either a rat CD4 or human IgG1 Fc tag coupled to either aC- or N-terminal FBG domain as described below.

Protein Expression Constructs

All synthetic DNA constructs for antigen expression were synthesised andsequence confirmed by Genscript (Piscataway, USA). FBG domains werecloned into the mammalian expression vectors pBIOCAM4 or BIOCAM5, whichfuse the expressed domains with either a rat Cd4 (domains 3 and 4) tag(Chapple et al, 2006) or a human IgG1 Fc tag (Falk et al, 2012)respectively. The vectors were modified from the pCMV/myc/ER plasmid(Invitrogen) (Falk et al, 2012), which contains an endoplasmic reticulum(ER) signal sequence derived from the mouse VH chain, for secretion ofexpressed proteins. For all constructs which resulted in an N-terminalFBG (e.g. FBG-Fc-His or FBG-rCd4-His) the digested PCR products wereligated with Ncol/NotI cut pBIOCAM4 or pBIOCAM5 vectors. For allconstructs which resulted in a C-terminal FBG (e.g. Fc-His-FBG orrCd4-His-FBG), digested PCR products were ligated with BamHI/HindIII cutpBIOCAM4 or pBIOCAM5 vectors. The primers used to amplify the FBGdomains are listed in FIG. 25. All constructs were sequence confirmed.To facilitate ELISA screening, an insert encoding a His-tag (primers2574 and 2575) was cloned between the BamHI and HindIII sites (replacingthe His-FLAG tag) for the expression plasmid with a FBG-X (N-terminalFBG) fusion. Full length tenascin C was cloned directly from theGenscript pUC57 plasmid by digestion with BstXI and BamHI and clonedinto the BstXI/BamHI cut expression vector pFBG-Fc-His6. To createHis-FBG constructs, primers were designed to PCR from an rCd4-His-FBGexpression plasmid and the PCR product, encoding His-FBG, was digestedwith XhoI and HindIII and cloned into the XhoI/HindIII digestedpBIOCAM5.

Protein Expression and Cell Culture

Transfection quality plasmid DNA was prepared using the Machery NagelNucleobond Xtra Midi kit (740410.50, Fisher Scientific, UK). HEK293Fsuspension cells and Freestyle media, for antigen and antibodyexpression, and RPMI media were from Life Technologies (Paisley, UK).Transfection of HEK293F cells was carried out as described previously(Chapple et al, 2006).

Protein Purification and QC

Protein affinity purification employed either Ni-NTA agarose orimmobilised recombinant protein A resin.

For purification of His-tagged proteins, culture supernatants were mixedwith Ni-NTA agarose (1018240, Qiagen, Crawley, UK) for 1 h and the resintransferred to Proteus 1-step midi spin columns (Generon, UK) forcentrifugation (200×g, 2 min). Unbound proteins were washed out withphosphate buffered saline (PBS) supplemented with 20 mM imidazole (pH8). Bound proteins were eluted in fractions through addition of 300 mMimidazole in PBS (pH 8) and column centrifugation (200×g, 2 min). Pooledfractions containing eluted protein were placed in Gebaflex Mididialysis tubes (Generon D010; molecular weight cut-off 3.5 kDa) anddialysed against PBS.

Fc-tagged proteins and antibodies expressed as human IgG4 were purifiedusing protein A sepharose (PC-A25, Generon, Maidenhead, UK). Culturesupernatants were clarified by centrifugation (2500×g, 15 min) and mixedwith protein A sepharose overnight at 4° C. before transfer of the resinto Proteus 1-step midi spin columns (Generon, UK). Columns werecentrifuged (200×g, 2 min) and washed with PBS to remove unboundprotein. Fc-tagged or IgG4 proteins were eluted in fractions from theprotein A with 0.2 M glycine (pH 2.8) into Tris-HCl (pH 8) bycentrifugation (200×g, 2 min). Eluted fractions were pooled and dialysedagainst PBS in Gebaflex Maxi dialysis tubes (Generon D045; molecularweight cut-off 8 kDa).

Proteins were analysed for purity and concentration by SDS-PAGE (4-12%gel) and spectrophotometry (OD280 using theoretical extinctioncoefficient). Where purified proteins were used in cell-based assays theendotoxin content was first determined by limulus amoebocyte lysatechromogenic endotoxin assay (Pierce). Proteins were not used ifendotoxin levels exceeded 1 endotoxin unit per milligram (i.e. 1 EU/mg).

Example 2—Isolation of Primary Anti-FBG Antibodies

Antibody Phage Display

Antibodies against tenascin-C FBG domain were isolated using the IontasLtd proprietary human antibody phage display library, which wasconstructed using DNA isolated from 43 human lymphocyte donors.Selections, phage rescues and subcloning into pSANG10 (Martin et al,2006) were all performed as described previously (Schofield et al, 2007)using techniques that are well known in the art.

Two rounds of panning selections were performed on immobilised TNC FBGfused to human IgG1 Fc or rCd4 at either the N terminus of the fusionpartner (e.g. FBG-Fc, FBG-rCd4) or at the C terminus (Fc-FBG, rCd4-FBG).Phage antibody libraries containing either kappa (κ) or lambda (Δ)variable light chains (V_(L)) were panned separately to facilitate latersub-cloning to Fab expression vectors containing either constant light(C_(L)) kappa (κ) or lambda (Δ) chains.

Polyclonal phage populations were prepared from the selected populationsand were tested in ELISA (polyclonal phage ELISA) using ELISA platescoated with TNC FBG antigen or appropriate fusion partner (Fc or rCd4).After incubation with phage, plates were washed, and bound phagedetected using peroxidase-conjugated anti-M13 antibodies. FIG. 3 showsenrichment of antigen-specific binders between rounds 1 and 2 ofselection and a greater proportion of FBG binders compared to anti-Fc or-rCd4 phage in the round 2 output populations, indicating that theselections were successful.

Confirmation of scFv Binding to Antigen and Cross-Reactivity Assay byELISA

Round 2 selection outputs were expressed as individual scFv clones toconfirm antigen recognition in ELISA binding assays. Output populationswere sub-cloned into the bacterial expression vector pSANG10 (Martin etal, 2006), transformed into E. coli BL21 (DE3), and individualtransformants were induced in 96-well plates as described previously(Schofield et al, 2007). E. coli supernatants were collected and assayedfor binding of scFv to TNC FBG using DELFIA-based ELISA, usingeuropium-labelled anti-FLAG detection antibodies.

Results for initial ELISAs are summarised in Table 1.

TABLE 1 Monoclonal scFv ELISA. Values indicate number of clones bindingto the relevant immobilised selection antigen. FBG Binders No. Tag ELISASignal (Fluorescence Units; FU) Selection (ID) screened binders ≥1,000≥10,000 ≥100,000 λ FBG-rCd4 (145) 95 0 0 0 0 λ FBG-Fc (146) 95 3 0 0 0 λrCd4-FBG (147) 95 0 14 4 0 λ Fc-FBG (148) 95 1 13 5 1 κ FBG-rCd4 (150)95 0 20 8 1 κ FBG-Fc (151) 95 0 2 1 0 κ rCd4-FBG (152) 95 8 12 4 0 κFc-FBG (153) 95 8 10 2 0 λ + κ FBG-rCd4, Fc 95 0 6 3 1 λ + κ rCD4-FBG,Fc 95 0 2 1 0 Total 79 28 3

The most successful selections with the A library were based on panningagainst the antigens rCd4-FBG and Fc-FBG (selections 147 and 148). Forthe K library, the most successful selections were obtained with theantigens FBG-rCd4 (150), rCd4-FBG (152) and Fc-FBG (153). The 79positive clones from this ELISA screen were selected for furtheranalysis.

Cross-reactivity ELISA showed that 67/79 (85%) of anti-human FBG scFvwere cross-reactive to mouse TNC FBG. DNA sequence analysis of theanti-FBG scFv indicated excellent sequence diversity. For example,selections 147 and 148 from the V_(L) A library contained 92% uniquevariable heavy (V_(H)) complementarity determining region 3 (CDR3)sequences, and selections 150, 152 and 153 from the V_(L) K librarycontained 67%, 91% and 100% unique variable V_(H) CDR3 sequences,respectively.

A further 1425 clones isolated from the most effective selections werescreened by ELISA and this resulted in the identification of anadditional 401 scFv with FBG-binding specificity (Table 2). Theseclones, together with the 79 scFv identified in initial ELISAs werechosen for further evaluation.

TABLE 2 Focused monoclonal scFv ELISA of the most effective selectionoutputs. No. Hits Tag FBG Selection (ID) screened (≥5,000 FU) bindersbinders λ rCd4-FBG (147) 285 66 0 66 λ Fc-FBG (148) 285 60 0 60 κFBG-rCd4 (150) 285 86 0 86 κ rCd4-FBG (152) 285 144 2 142 κ Fc-FBG (153)285 94 47 47 Total 1425 450 49 401

The 1425 clones were further tested in a specificity ELISA in which eachscFv was tested for binding to human Tenascin R FBG and also to human,mouse, rat and dog TNC FBG. Clones were ranked according to the ELISAsignal obtained for binding to Tenascin C divided by the signal forTenascin R FBG binding. The top 250 clones with a ratio above 50 weretaken for subcloning and further analysis.

Example 3—Screening of Primary Anti-FBG Antibodies in a Functional Assay

Anti-FBG scFv were reformatted either as bivalent scFv-Fc or asmonomeric Fabs for evaluation of their activity as inhibitors ofFBG-evoked signalling in a whole cell assay system.

The top 50 anti-FBG scFv, ranked by primary ELISA signal, for each ofthe selections 147, 148, 150, 152 and 153 were sub-cloned into themammalian expression plasmid pBIOCAM5 (Falk et al, 2012) as individualselection populations and expressed by transient transfection in HEK293Fcells (Chapple et al, 2006). For Fab expression, pooled A or K scFvvariable heavy (V_(H)) and variable light (V_(L)) inserts were clonedinto a dual promoter Fab expression vector (pFab-dual-κ or pFab-dual-λ,depending on the light chain germ-line) using a proprietary Iontas Ltdprotocol. Culture supernatants were screened for activity in the THP-1cell assay and selected scFV-Fc and Fab hits were affinity purified forre-assaying and confirmation of inhibitory activity.

THP1-Blue™ Reporter Cell Assay

Tenascin-C has been shown to elicit the generation of cytokines ininflammatory cells and fibroblasts by interaction of the FBG domain withcellular TLR4 (Midwood et al, 2009). The receptor signalling cascadeleading to generation of inflammatory cytokines such as TNFa, IL-8 andIL-6 involves activation of the transcription factor NF-κB. This processcan be studied in ‘reporter’ cell lines modified to respond to NF-κBactivation with generation of an easily measured protein signal. TheTHP1-Blue™ reporter cell line (InvivoGen; Toulouse, France) is derivedfrom the human THP-1 monocyte cell line and stably expresses anNF_KB-inducible secreted alkaline phosphatase (SEAP) reporter construct.These cells also constitutively express cell surface TLR4, which enablesthe signalling activity of TNC FBG fusion proteins to be readilymeasured using colorimetric or fluorimetric quantitation of SEAP inculture supernatants using medium- to high-throughput assay methods.

Activity at low FBG concentrations is critical to the success of anyscreening assay; if the concentrations of FBG required to produce arobust increase in the reporter protein are too high then the expressionlevels and concentrations of scFv, Fc-ScFv or Fab constructs required tofully inhibit any such signal would be unacceptable for a screen. Fc-FBGproduces a robust SEAP signal at low nM levels in this cell assay(CD4-FBG did not produce a response in this concentration range).

THP1-Blue™ cells were cultured and passaged in supplemented RPMI mediaaccording to supplier's protocols(http://www.invivogen.com/PDF/THP1_Blue_NF_kB_TDS.pdf), except thatcells were grown in ultra-low attachment T75 flasks. For assays,THP1-Blue™ cells were added to 96-well tissue culture plates (100,000cells/well) containing Fc-FBG (3 or 10 nM) in RPMI medium in a totalvolume of 170 μl. Culture supernatants containing expressed scFv-Fc orFab, or affinity purified antibody in PBS, was added in a volume of 30μl and cells were incubated for 18 h at 37° C. Supernatants wereharvested and assayed for either SEAP using the Attophos AP fluorimetricquantitation system (S1000; Promega) or IL-8 content using the DuoSetELISA development system (DY208; R&D Systems, UK) according to thesupplier's instructions. Data were plotted and curves fitted using Prismsoftware (GraphPad).

Screening of anti-FBG antibodies as HEK293F culture supernatantshighlighted putative inhibitors of Fc-His-FBG evoked signalling inTHP1-Blue™ cells of which 9 were confirmed when re-assayed as purifiedscFv-Fc (FIG. 4A) or Fab (FIG. 4B). Fc-His-FBG is key to having thepotecy assays work. Monomeric FBG does not elicit any cytokine responsein THP-1Blue and human cells.

Example 4—Functional Characterisation of Primary Anti-FBG Antibodies

ELISA Cross-Reactivity Assays

The panel of 9 human FBG signalling inhibitors identified in theTHP1-Blue™ functional assay was evaluated by ELISA for cross-reactivityto rat, mouse, and dog FBG. Binding to the human tenascin-R FBGhomologue was also determined. Assay wells were coated with human, rat,mouse, and dog TNC FBG-rCD4, or human TNR FBG-rCd4 fusion proteins andbinding of Fabs was detected using anti-kappa or anti-lambda mAbfollowed by Europium-conjugated anti-mouse mAb. ELISA results revealedthat 4 Fabs displayed good cross-reactivity to other mammalianhomologues of human TNC FBG, with lower apparent binding to human TNRFBG (FIG. 5). These were:

Fab 2A5 (VH SEQ ID NO: 4; VL SEQ ID NO: 8),

Fab B12 (VH SEQ ID NO: 12; VL SEQ ID NO: 15),

Fab D8 (VH SEQ ID NO: 19; VL SEQ ID NO: 21), and

Fab F3 (VH SEQ ID NO: 25; VL SEQ ID NO: 29).

Fabs that showed poor species cross-reactivity to TNC-FBG were notconsidered further.

Determination of Binding Affinity by Surface Plasmon Resonance

The affinity and association and dissociation kinetics of selected Fabsfor binding to the human, rat and mouse TNC FBG, and human TNR FBG weremeasured by surface plasmon resonance (SPR) at 25° C. Experiments wereperformed using a BIAcore T100 instrument with CM5 sensor chip accordingto the protocol provided with the Human Fab Capture Kit (GE,28-9583-25). Varying concentrations of rCd4-FBG were injected into aflow-cell with immobilised Fab and a reference flow-cell. Afterreference signal subtraction, the data was fitted to a global 1:1 fitusing the T100 BIAevaluation software (FIGS. 6A-6D).

The calculated kinetic constants are shown in Table 3. The rank order ofaffinity of Fabs for human TNC FBG was B12 (110 pM)>D8 (8.49 nM)>2A5(11.4 nM)>F3 (27.4 nM). All Fabs displayed low nanomolar affinity forrodent TNC FBG, and affinities for human TNR FBG were typically greaterthan 60-fold lower than human TNR FBG.

Inhibitory Potency Assays

The potency of purified Fabs for neutralisation of huFc-His-FBG activitywas determined in the THP1-Blue™ assay, using measures of TLR4-mediatedsecreted alkaline phosphatase and IL-8 cytokine production. Assays wereconducted as described in Example 2, except that purified Fabs wereadded to assay wells at a range of concentrations (0.3-100 nM) to enablecalculation of IC₅₀ values using Prism software (GraphPad).

TABLE 3 Anti-FBG Fab binding kinetic data determined by surface plasmonresonance (SPR) spectroscopy. Kinetics K_(D) K_(a) K_(d) Steady Fab FBG(nM) (M⁻¹s⁻¹) × 10⁵ (S⁻¹) × 10⁻⁴ State 2A5 Hu TNC 11.4 4.96 56.3 N/A MuTNC 78.6 4.41 346.5 N/A Hu TNR 757 2.49 1888.4 706   B12 Hu TNC 0.11126.62 3.0 N/A Mu TNC 13 52.15 675.5 18.7 Rat TNC 7.9 94.59 747.9 N/A HuTNR 33.9 13.96 472.5 36.1 D8 Hu TNC 8.49 15.41 130.9 N/A Mu TNC 48.414.78 716.1 41.2 Hu TNR 1026 5.55 5696.0 913   F3 Hu TNC 27.4 1.26 34.6N/A Mu TNC 70.6 0.91 64.2 N/A Hu TNR Off rate too rapid to determine1808 K_(D), equilibrium dissociation constant; K_(a), associationconstant; K_(d), dissociation constant

All antibodies displayed concentration-related inhibition ofFc-His-HuFBG-evoked alkaline phosphatase (FIG. 7) and IL-8 production(FIG. 8). The rank order of potency (IC₅₀) for inhibition of alkalinephosphatase inhibition by anti-FBG Fabs was B12 (1.7 nM)>D8 (7.2 nM)>F3(8.4 nM)>2A5 (20.6 nM), and the potency (IC₅₀) ranking was similar forinhibition of IL-8 release: B12 (6.9 nM)>F3 (13.8 nM)>D8 (14.9 nM)>2A5(28.5 nM).

Example 5—Generation and Isolation of Optimised Antibodies to huTNC FBGDomain

Affinity Maturation by Targeted CDR Mutagenesis

Anti-FBG antibodies 2A5, B12, and F3 were selected for affinitymaturation. Targeted CDR mutagenesis was carried out by randomising VHand VL CDR3 residues in blocks of 6 amino acids using Kunkel mutagenesis(Fellouse and Sidhu, 2007; Kunkel et al., 1987; Sidhu and Weiss, 2004).Due to the longer VH CDR3s (10-16 residues) for the given clonesrandomisation was done in three overlapping blocks and the VL CDR3s (9residues) were randomised in two overlapping blocks (FIG. 9A).Randomisations were carried out using NNS (N=A/G/C/T and S=G/C)degenerate primers that could encode any of the 20 amino acids (and onlya single amber stop codon) at a given position from 32 codoncombinations. Oligonucleotides used in the mutagenesis are provided inFIG. 9B. Thus, 15 libraries (3 libraries per VH and 2 libraries per VLfor 3 antibodies) were created initially and all libraries except for F3VL 3.1 and F3 VL 3.2 were large enough (Table 4) to cover thetheoretical diversity arising from randomising 6 residues with an NNSprimer (32⁶=1.1×10⁹). The CDR3 libraries were combined during the rescueprocess and this resulted in a combined mutant VH library and a combinedmutant VL library for each of the parental antibody clones, giving 6libraries in total.

TABLE 4 Estimated sizes of the CDR3 randomised libraries Library Sublibrary Size Combined size 2A5 VH 2A5 VH 3.1 2.0 × 10⁹ 7.2 × 10⁹ 2A5 VH3.2 2.6 × 10⁹ 2A5 VH 3.3 2.6 × 10⁹ 2A5 VL 2A5 VL 3.1 4.0 × 10⁹ 6.5 × 10⁹2A5 VL 3.2 2.5 × 10⁹ B12 VH B12 VH 3.1 1.8 × 10⁹ 6.1 × 10⁹ B12 VH 3.21.6 × 10⁹ B12 VH 3.3 1.7 × 10⁹ B12 VL B12 VL 3.1 2.6 × 10⁹ 7.7 × 10⁹ B12VL 3.2 5.1 × 10⁹ F3 VH F3 VH 3.1 6.0 × 10⁹ 1.6 × 10⁹ F3 VH 3.2 4.6 × 10⁹F3 VH 3.3 6.3 × 10⁹ F3 VL F3 VL 3.1 2.1 × 10⁹ 5.7 × 10⁹ F3 VL 3.2 3.6 ×10⁹

High Stringency Phage Display Selections

Phage-antibody selections on streptavidin Dynabeads were performed asdescribed previously (Dyson et al, 2011). Multiple rounds ofsolution-phase selections were carried out on biotinylated rCd4-His-FBGto enrich for affinity improved clones. The optimum antigenconcentrations for each round were determined empirically by selectingagainst a range of antigen concentrations and comparing the outputnumbers with a no-antigen control. The stringency of selection wasincreased by reducing the amount of antigen used in each round. Nofurther rounds of selection were carried out after the selection window(the fold difference between phage titres from selection outputs and noantigen control) dropped below 10. Hence, three rounds of selection(FIG. 10A) were carried out on biotinylated human rCd4-His-FBG for alllibraries except B12 which was subjected to a fourth round of selectiondue to the large selection windows observed at round 3. All librarieswere subjected to deselection against streptavidin beads and tenascin-R(100 nM for rounds 1 to 3 and 1 nM for round 4) at each round ofselection to avoid unwanted cross reactivity to streptavidin ortenascin-R. In addition, a hybrid selection strategy in which the humanand mouse antigens were alternated between rounds of selection (FIG.10B) was performed for the B12 randomised libraries only. The reason forperforming this extra selection on the B12 libraries was the largedifference in affinity observed for the B12 parental antibody binding tohuman and mouse rCd4-his-FBG. This difference was not as pronounced forthe 2A5 (6.9-fold) or F3 (2.6-fold) parental antibodies. Furthermore, anadditional round of selection was carried out to select for antibodyclones with superior off-rates. In off-rate selections, phage wereallowed to bind to the biotinylated antigen (1 nM in this case), and alarge excess of non-biotinylated antigen (500 nM) was subsequently addedto the reaction and incubated for 20 h or 40 h. The non-biotinylatedantigen serves as a competitor and captures the phage antibodies thatdissociate from the biotinylated antigen, i.e. only the antibodies withlonger off-rates will be recovered at the end of the selection (Hawkinset al., 1992; Zahnd et al., 2010). The output phage titres for eachround of selection together with calculated selection windows are shownin Tables 5a-c.

The selected populations were sub-cloned into the bacterial expressionvector pSANG10 (Martin et al, 2006), transformed into E. coli BL21(DE3),and individual transformants picked (46 per selection) for ELISA andHTRF analyses in order to identify clones with improved binding to mouseFBG and human FBG respectively.

TABLE 5a Selection output titres. Round 1 selections. Phage outputtitres were determined as described previously (Schofield et al, 2007)Selection Selection CDR3 window window randomised 10 nM 1 nM 0 nM for 10nM for 1 nM libraries Selection Selection Selection selection selection2A5 VH 7 × 10⁷ 2.9 × 10⁷ 1 × 10⁵ 700 290 2A5 VL 3 × 10⁷ 1.7 × 10⁷ 5 ×10⁴ 600 340 B12 VH 6 × 10⁷ 2.6 × 10⁷ 1 × 10⁵ 600 260 B12 VL 6 × 10⁷  5 ×10⁷ 2 × 10⁵ 300 250 F3 VH >1 × 10⁸   8 × 10⁷ 2 × 10⁴ 5000 4000 F3 VL 5 ×10⁷ 1.2 × 10⁷ 9 × 10⁴ 555 133

TABLE 5b Selection output titres. Round 2 selections. Phage outputtitres were determined as described previously (Schofield et al, 2007)Selection CDR3 window for Selection window randomised 200 pM 50 pM O nM200 pM for 50 pM libraries Selection Selection Selection selectionselection 2A5 VH  7 × 10⁷ 3.8 × 10⁷ 5 × 10⁴ 1400 760 2A5 VL 1.4 × 10⁷  6× 10⁶ 1 × 10⁴ 1400 600 B12 VH  1 × 10⁸ 6.75 × 10⁷  2 × 10⁴ 5000 3375 B12VL 1.2 × 10⁸ 8.1 × 10⁷ 4 × 10⁴ 3000 2025 F3 VH 1.1 × 10⁸ 9.5 × 10⁷ 4 ×10⁴ 2750 2375 F3 VL  7 × 10⁷ 1.2 × 10⁷ 1.2 × 10⁵  583 100 B12 VH on mu 7 × 10⁶ 2 × 10⁴ 350 TNC FBG B12 VL on mu 7.5 × 10⁶ 4 × 10⁴ 187 TNC FBG

TABLE 5c Selection output titres. Round 3 selections. Phage outputtitres were determined as described previously (Schofield et al, 2007)Selection Selection CDR3 window window for randomised 5 pM 1 pM 0 nM for5 pM 1 pM libraries Selection Selection Selection selection selection2A5 VH   6 × 10⁶  1 × 10⁶ <1 × 10⁵ 60 10 2A5 VL 1.4 × 10⁶ <1 × 10⁵  2 ×10⁵ 7 <1 B12 VH 1.5 × 10⁷  4 × 10⁶ <1 × 10⁵ >150 >40 B12 VL 2.7 × 10⁷3.5 × 10⁶  <1 × 10⁵ >270 >35 F3 VH 3.5 × 10⁶  4 × 10⁵ <1 × 10⁵ >35 >4 F3VL   6 × 10⁵ <1 × 10⁵  2 × 10⁵ 3 <1 Selection Selection Hybridselections window window for on B12 libraries 20 pM 5 pM 0 pM for 20 pM5 pM (Hu-mu-hu) Selection Selection Selection selection selection B12 VH  1 × 10⁸ 7.7 × 10⁶ <1 × 10⁵ >1000 >77 B12 VL 1.3 × 10⁸ 1.8 × 10⁷ <1 ×10⁵ >1300 >78

ELISA Screen

An anti-FLAG capture ELISA was performed to screen for clones that hadan improved affinity for mouse FBG binding compared with the parentalantibodies.

E. coli BL21 (DE3) clones harbouring scFv pSANG10 expression plasmidswere induced in 96-well plates with auto-induction media as describedpreviously (Schofield et al, 2007). E. coli supernatants were harvestedfor ELISA assays. ELISA used the DELFIA (dissociation enhancedlanthanide fluorescent immunoassay) system with Europium-labelledanti-FLAG antibody (Sigma, Aldrich, UK). Black immunosorb plates (Nunc)were coated overnight with anti-FLAG M2 antibody (Sigma, F3165, 5 μg/mlin PBS, 50 μl per well), in wells blocked by the addition of 2% milkpowder, PBS (PBS-M, 300 μl per well). Plates were washed three timeswith PBS-T (PBS, 0.1% Tween-20) and three times with PBS followed by theaddition of a 1:2 dilution of 96-well auto-induction culturesupernatants containing expressed scFv in PBS-M (50 μl per well). Theplates were incubated for 1 h, washed as above and biotinylated mouse orhuman rCd4-His-FBG (5 μg/ml in PBS-M, 50 μl) added to each well. Plateswere incubated for a further 1 h, washed and Strepravidin-Eu added(Perkin Elmer, 1 μg/ml, PBS-M, 50 μl), incubated for 30 min, washed andDELFIA enhancement solution added (50 μl) and plates read on a PerkinElmer Fusion plate reader (excitation=320 nm, emission 620 nm). Theformat of the assay is shown in FIG. 11.

In this assay differences in scFv expression level are normalisedbecause the expression levels of scFv in auto-induction culturessaturate the anti-FLAG coated wells. Therefore, the signals obtained inthe assay reflect the amount of biotinylated rCd4-His-FBG bound afterwashing, which will be a function of the off-rate of that clone formouse or human FBG. ELISA screening of the selection output from the 2A5and B12 sub-libraries revealed clones with significantly improvedbinding to mouse TNC FBG.

HTRF Screen

An HTRF-based competition assay was developed to screen for antibodyvariants with improved binding to human TNC FBG.

All samples and reagents were prepared in assay buffer (50 mM NaPO₄,0.1% BSA, 0.4 M KF, pH 7.0) at 4×the stated concentration. 5 μl of eachreagent was subsequently added to low volume 384-well assay plates(Greiner, 784075) to give a final reaction volume of 20 μl. IgGantibodies were labelled using the d2 labelling kit (CisBio, 62D2DPEA)as directed by the manufacturer. Streptavidin europium cryptate (CisBio,610SAKLA, Lot#25C) was used at a final concentration of 1.8 ng activemoiety (SA) per 20 μl reaction as recommended by the manufacturer.Biotinylated rCd4-His-FBG was prepared using EZ-link Sulfo-NHS-LC-Biotinreagent (Thermo Scientific, 21327) the extent of biotinylation wasquantified using biotinylation fluorescence quantitation kit (ThermoScientific, 46610). Where appropriate, supernatants containing scFv(prepared as described above for ELISA assays) were added to the384-well assay plate at a final dilution of 1/20 (i.e. ⅕ dilution inassay buffer followed by addition of 5 μl diluted sample to the 20 μlFRET assay). The concentrations of d2-labelled 2A5 IgG and B12 IgG usedfor screening were 15 nM and 1.25 nM respectively. Unless otherwisestated, biotinylated rCd4-His-FBG (biotin:protein ratio=1.8:1) waspresent at either 2.2 nM (in assays using the 2A5 IgG antibody) or 1 nM(in experiments using B12 IgG). Samples were incubated for approximately1 h at room temperature and the FRET signal was determined using a BMGPherastar instrument: excitation=320 nm; emission=620 nm and 665 nm;integration start time=60 μs; integration time=500 μs; 100 flashes perwell. For competition assays containing culture supernatant,biotinylated rCd4-His-FBG antigen was pre-incubated with streptavidineuropium cryptate for 45 min prior to addition of reagents to the assayplate. All FRET signals are presented as ΔR, where R=(E665/E620×104) andΔR=(Rsample−Rbackground fluorescence).

Culture supernatants containing unlabelled scFv clones from affinityselected mutant libraries were tested for inhibition of the interactionbetween FBG and the fluorophore-labelled parental IgG antibody. Whenused to screen the 2A5 variants, this approach yielded a high proportionof clones with improved inhibition relative to the parent (92% of VHCDR3 variants and 79% of VL CDR3 variants). In order to distinguishbetween the clones that fully inhibited the FRET signal, 2A5 variantswere subsequently screened for their ability to compete with B12 IgG.This was a more stringent screen given that the affinity of B12 forhuman FBG is approximately 100-fold stronger than that of 2A5(dissociation constants for these interactions, determined by surfaceplasmon resonance at 25° C. were 0.11 nM and 15 nM, respectively). Therelative ranking of clones exhibiting FRET signals within the usefulrange in both assays was broadly unchanged, indicating that they werecompeting for similar epitopes. Hence, all 2A5 and B12 scFv variantsfrom affinity maturation selections were screened for their ability toinhibit the binding of B12 IgG molecules to human TNC FBG. The parentalclones, expressed as scFvs in parallel with the affinity matured clones,were used as benchmarks (Table 6).

ScFv were sequenced and a panel of clones with unique VH or VL CDR3sequences was selected for further study in human IgG4 format, based ontheir binding to mouse and human TNC FBG in the ELISA and HTRF assays,respectively. The chosen variants of antibody 2A5 displayed ≥10-foldimprovement in binding to the mouse FBG and an inhibition of ≥90% (VHCDR3 variants) or 83% (VL CDR3 variants) in the HTRF assay.

TABLE 6 HTRF screen for clones with improved affinity for humanrCD4-FBG. Total % inhibition of FRET signal % inhibition CDR3 Selectionclones 0- 25- 51- 76- 86- 91- by parent scFv Library type tested 25% 50%75% 85% 90% 95% ≥96% 2A5 B12 2A5 VH 1 pM 46 3 4 4 15 13 7 0 29 90 2A5 VHOff-rate 46 3 0 11 11 17 3 1 29 90 2A5 VL 5 pM 46 3 2 7 19 14 1 0 21 832A5 VL Off-rate 46 10 11 10 5 10 0 0 21 83 B12 VH 100 fM   46 6 2 3 8 56 16 19 86 B12 VH Hybrid 46 3 3 5 5 3 9 18 19 86 5 pM

Variants of antibody B12 showed ≥4-fold improvement for mouse FBGbinding, and 91% inhibition of HTRF signal. In total, 31 clones fittingthese criteria with unique CDR3 sequences were identified (Table 7).

TABLE 7 Heavy or light chain CDR3 sequences of clonesidentified with improved binding to mouse andhuman TNC FBG and chosen for conversion tohuman IgG format for further study. Library Clone name CDR sequenceB12 VH 165_13_B1 VMSSMEDAFDI SEQ ID NO: 30 165_13_B6 GQKGEGDTFDISEQ ID NO: 32 165_13_D1 GTRGEGDTFDI SEQ ID NO: 34 165_13_C3 SYQSDEDAFDISEQ ID NO: 36 165_13_D4 GTVGEGDTFDI SEQ ID NO: 38 165_13_A4 DKYPVLDTFDISEQ ID NO: 40 165_13_B3 ALARGHDTFDI SEQ ID NO: 42 165_13_E1 DISAVMDVPQTSEQ ID NO: 44 180_11_F5 VMRTGLDTFDI SEQ ID NO: 46 2A5 VH 160_01_E3QRYVWEALTY SEQ ID NO: 48 160_01_D6 AQADPHLFTY SEQ ID NO: 50 160_01_H4GRFVWEALTY SEQ ID NO: 52 160_01_A4 AQKETLGNAI SEQ ID NO: 54 160_01_F1AQSPWSGMTY SEQ ID NO: 56 160_01_G2 YTLDNMALTY SEQ ID NO: 58 161_01_F6AQKENIANRH SEQ ID NO: 60 (160_01_F6) 161_01_A12 AQPTALANTY SEQ ID NO: 62161_01_C09 AQLPYLAQTY SEQ ID NO: 64 161_01_H10 AQPVWAPGTY SEQ ID NO: 66161_01_C11 AQKEWLPDVT SEQ ID NO: 68 162_02_D3 AQIHPLGLTY SEQ ID NO: 702A5 VL 162_02_C6 QNQYAGPWT SEQ ID NO: 72 162_02_H5 QNQYTGPWTSEQ ID NO: 74 162_02_F3 QNQYRGPWT SEQ ID NO: 76 162_02_C1 LHHYRAPWTSEQ ID NO: 78 162_02_C2 MHHYRAPWT SEQ ID NO: 80 162_02_F4 MHHYRSPWTSEQ ID NO: 82 162_02_03 MQHYDGPWT SEQ ID NO: 84 162_02_E11 LHHYRSPTWTSEQ ID NO: 86 162_02_E11 LHHYRSPWT SEQ ID NO: 135 163_02_A12 LHHYREPWTSEQ ID NO: 88 163_02_D11 LHHYKSPWT SEQ ID NO: 90

These are heavy or light chain sequences of antibody clones that bind tohuman and mouse TNC FBG and thus have potential utility in the methods,uses, compositions and compounds of the present invention. For example,antibodies that bind TNF FBG having these CDR3 sequences may be usefulin identifying, inhibiting the function of, detecting and purifying TNCor TNC FBG.

Conversion to IgG4 Format and Determination of Binding Kinetics

The 31 scFv of interest were sub-cloned into a human IgG4 expressionvector for generation of antibodies as human IgG4 with ahinge-stabilising mutation (S241P; Angal et al, 1993). IgG4 antibodieswere transiently expressed in HEK-293F cells and culture supernatantswere screened using surface plasmon resonance spectroscopy for rankingof their off-rates for binding to human and mouse TNC FBG, and human TNRFBG. Briefly, surface plasmon resonance (SPR) experiments were performedusing a BIAcore T100 instrument and followed the protocol according tothe Human antibody capture kit protocol (GE, BR-1008-39). For off-ratescreening, 10,000 response units (RU) of anti-human Fc IgG (GE,BR-1008-39) was immobilised on flow-cells (FC1 and FC2) of a Series 5CM5 dextran sensor chip (BR-1005-30) using EDC/NHS cross-linkingchemistry according to the amine coupling kit protocol (GE, BR-1000-50).Culture supernatants containing expressed IgG4 were diluted 1:2 with2×PBS-T and injected into FC2 (flowrate 5 μl/min, 60s contact time) toenable antibody capture at 25° C. Antibody capture levels ranged from308 to 1975 RU depending on the expression level of the antibody in thesupernatant. A fixed concentration of antigen (15 nM of human and mouseTNC rCd4-His-FBG and 100 nM of human TNR rCd4-His-FBG) was injected witha flow-path via FC 1 (reference flow cell) and FC 2 (antibody captureflow cell), with a flow rate of 30 μl/min, and the association anddissociation phases measured over 1 and 5 min time periods,respectively. Regeneration of the binding surface employed 3M MgCl₂ with30s contact time. Off rates were determined by reference cellsubtraction and fitting the sensogram experimental data assuming a 1:1interaction using BIAevaluation software (GE, BR-1005-97). Results ofthe off-rate screen are summarised in Table 8.

TABLE 8 Surface plasmon resonance screen for ranking of human IgG4anti-FBG off-rates kd (s⁻¹ × 10⁻⁴) for rCD4-His-FBG Clone name Human TNCFBG Mouse TNC FBG Human TNR FBG 165_13_B1 0.015 0.017 390 165_13_B60.056 0.069 37 165_13_D1 0.0014 0.039 43 165_13_03 0.00095 0.033 120165_13_D4 0.0062 0.037 40 165_13_A4 8.72 79.7 nd 165_13_B3 0.014 300 nd165_13_E1 0.014 577 nd 180_11_F5 0.26 10000 nd 160_01_E3 0 558.8 nd160_01_D6 0.105 558.8 nd 160_01_H4 0.16 170.8 nd 160_01_A4 0.067 0.059110 160_01_F1 0.04 1540000 nd 160_01_G2 0.125 0.139 10 161_01_F6 0.02817.1 25 (160_01_F6) 161_01_A12 0.013 0.043 42 161_01_C09 0.00117 0.00232.9 161_01_H10 0.25 0.019 91 161_01_C11 0.0022 nd nd 162_02 _D3 0.00390.0106 64 162_02 _C6 0.053 2.4 280 162_02 _H5 0.00043 1.67 820 162_02_F3 0.00083 3.3 880 162_02_C1 0.00093 16 27000000 162_02_C2 0.115 17535000 162_02_F4 0.0059 10 151000 162_02_C3 0.0149 20 6350 162_02_E110.011 12 10110000 163_02_A12 0.0032 9.4 288000 163_02_D11 0.0032 9.822090000 2A5 parent 91 590000 2720 B12 parent 1.5 300 1001

Clones were ranked according to low off-rate for human and mouse TNCrCd4-His-FBG, and high-off rate for human TNR rCd4-His-FBG. The 3highest-ranking antibodies from each library were prioritised for moredetailed kinetic analysis as purified IgG4. These clones are shown inTables 9, 10 and 11.

TABLE 9 Heavy chain CDR3 amino acid sequences ofB12 mutants with improved FBG binding off-rate characteristics CloneVH CDR3 B12 parent DISAVPDTFDI SEQ ID NO: 11 165_13_B1 VM S SME D A FDISEQ ID NO: 30 165_13_D1 GTRGEG DTFDI SEQ ID NO: 34 165_13_03 SYQSDE D AFDI SEQ ID NO: 36

TABLE 10 Heavy chain CDR3 amino acid sequences of2A5 mutants with improved FBG binding off-rate characteristics CloneVH CDR3 2A5 parent AQKETYALTY SEQ ID NO: 3 160_01_A4 AQKET LGNAISEQ ID NO: 54 161_01_H10 AQ PVWAPG TY SEQ ID NO: 66 162_02_D3 AQ IHPLGLTY SEQ ID NO: 70

TABLE 11 Light chain CDR3 amino acid sequences of2A5 mutants with improved FBG binding off-rate characteristics. CloneVL CDR3 2A5 parent QQSYSTPWT SEQ ID NO: 7 162_02_F3 Q NQ Y RG PWTSEQ ID NO: 76 163_02_A12 LHH Y RE PWT SEQ ID NO: 88 163_02_D11 LHH Y KSPWT SEQ ID NO: 90

Detailed kinetic parameters were evaluated for the 9 prioritised IgG4antibodies. Binding characteristics were determined for interaction withhuman, rat and dog TNC rCD4-His-FBG, and human TNR rCD4-His-FBG. Kineticassays followed essentially the same protocols as for the off-ratedeterminations described above, with some modifications as follows. Toimprove the accuracy of kinetic parameter determination, anti-human FcIgG was immobilised at lower levels (2229 RU), resulting in acorresponding reduction in the amount of anti-FBG IgG4 captured.Purified anti-FBG IgG4 was diluted to a concentration of 3.5 nM in PBS,pH 7.4, 0.05% Tween-20 and injected into FC2 at a flow rate of 10μl/min, 60s contact time. This typically resulted in an average of 80 RUof antibody captured (range: 55 RU to 90 RU). Antigens were prepared bydoubling dilution in PBS, pH 7.4, 0.05% Tween-20 (highest concentration100 nM except mouse rCD4-His-FBG which was 7 nM). Assays were performedat 37° C. (30 μl/min, 120s contact time; mouse rCD4-His-FBGFBG 10μl/min, 60s contact time), with both the flow cell and injection chamberequilibrated to this temperature. As before, kinetic parameters weredetermined by reference cell subtraction and fitting the sensogramexperimental data assuming a 1:1 interaction using BIAevaluationsoftware (GE, BR-1005-97).

All nine antibodies displayed improved binding to mouse TNC FBG domaincompared to the non-affinity matured parent clones, and antibodies165_13_61, 165_13_03, and 160_01_A4 exhibited sub-nanomolar K_(d) valuesfor binding to human TNC FBG, with >70-fold lower affinity to the humanTNR FBG analogue (Table 12).

TABLE 12 Anti-FBG IgG4 binding kinetic data determined by surfaceplasmon resonance at 37° C. K_(a) K_(d) Antibody rCD4-His-FBG K_(D)(M⁻¹s⁻¹) × (s⁻¹) × IgG4 Parent Species Tenascin (nM) 10⁴ 10⁻⁴ 2A5 2A5Human TNC 23.8 13.6 323 Mouse TNC 123 8.68 106.5 B12 B12 Human TNC 0.2447.1 11.2 Mouse TNC 4.5 30 13.8 165_13_B1 B12 Human TNC 0.26 72.7 18.8Mouse TNC 0.96 73.3 7.06 Rat TNC 2.20 31.1 68.4 Dog TNC 2.85 65.5 187Human TNR 94.4 12.2 1149 165_13_C3 B12 Human TNC 0.072 116 8.3 Mouse TNC0.46 97.2 4.45 Rat TNC 1.22 38.9 47.3 Dog TNC 1.80 59.7 108 Human TNR35.8 12.0 431 160_01_A4 2A5 Human TNC 0.21 23.5 5.0 Mouse TNC 1.23 11.81.46 Rat TNC 1.49 12.7 18.9 Dog TNC 0.094 19.0 1.8 Human TNR 15.2 2.639.9

Example 6—Inhibition of TNC FBG-Evoked Cytokine Production in PrimaryHuman PBMCs

The functional FBG neutralising activity of purified IgG4 antibodies165_13_61, 165_13_03, and 160_01_A4 was confirmed in an in vitro assayof FBG-evoked cytokine release in primary human PBMCs.

Peripheral blood mononuclear cell (PBMC) populations were isolated fromthree healthy human single donor buffy coat preparations by densitygradient centrifugation. Assays were carried out in 96-well plates in afinal volume of 200 μl, and the endotoxin content of all reagents andtest antibodies was confirmed to be within acceptable limits before use,determined using a limulus amoebocyte lysate (LAL) endotoxinquantitation kit (Pierce).

Freshly isolated PBMC samples (2×10⁵ cells/well) were cultured in thepresence of test antibodies (100 nM and 1 μM), control isotype antibody(Sigma 14639; 100 nM and 1 μM), dexamethasone (1 μM) or PBS control for1 h prior to submaximal stimulation with either bacteriallipopolysaccharide (LPS; E. coli 026:66; 100 ng/mL) or human Fc-His-FBG(200 nM). Control wells, in which LPS or Fc-His-FBG were replaced withan equal volume of PBS, contained test antibodies or dexamethasone.After incubation (24 h, 37° C.), culture supernatants were collected andstored at −80° C. Samples were thawed to room temperature before assayof supernatants for cytokine content. A 25 μl aliquot of eachsupernatant was diluted with an equal volume of RPMI medium (LifeTechnologies) and resulting samples were assayed in duplicate for IL-8and TNFα by Luminex analysis.

Incubation of PBMCs with 100 ng/mL LPS for 24 h resulted in IL-8 andTNFα production, which was not inhibited by exposure to either controlIgG4 antibody or the anti-FBG antibodies. In contrast, IL-8 and TNFarelease evoked by Fc-His-FBG was completely blocked by all testantibodies, but not control IgG4, confirming the potent and specificFBG-neutralising activity of the 3 affinity-matured antibodies165_13_61, 165_13_C3, and 160_01_A4 (FIGS. 12A, 12B).

Example 7—Anti-FBG IgG4 Binding to Citrullinated FBG

The binding affinity of antibody B12 to citrullinated FBG was determinedby surface plasmon resonance (SPR). B12 was expressed as a human IgG4with the hinge-stabilising 5241P mutation using the QMCF expressiontechnology (Icosagen, Estonia) and purified by protein A affinitychromatography (MabSelect Sure; GE Healthcare).

Citrullination of Human TNC FBG

Purified human His-FBG was citrullinated using either peptidylargininedeiminase 2 (PAD2; MQ-16.201-2.5, Modiquest, NL) or peptidylargininedeiminase 4 (PAD4; MQ-16.203-2.5, Modiquest, NL) according to thesupplier's instructions. Briefly, His-FBG was diluted to 1 mg/ml in thesupplied deimination buffer (0.1 M Tris-HCl pH 7.5, 10 mM CaCl₂), 5 mMdithiothreitol) and 250 μl mixed with 125 mU of either PAD2 or PAD4enzyme followed by incubation at 37° C. for 2 h. Citrullination wasconfirmed by amino acid analysis of the enzymatically-treated samples.Aliquots of His-FBG in deimination buffer were incubated for 2 h at 37°C. in the absence of added PAD enzyme, for use as non-citrullinatedcontrol protein. Citrullinated and unmodified His-FBG proteins were usedin SPR experiments as described below.

Surface Plasmon Resonance

SPR experiments were performed on a BIAcore 3000 instrument. Anti-humanIgG (GE Healthcare) was covalently coupled to the surface of a CM5sensor chip using amino coupling chemistry. The amount of the coupledanti-human IgG expressed in RU units varied between 6500-7000 (6.5-7.0ng/mm²). B12-hIgG4 (1-13 nM) was attached to the immobilised anti-humanIgG in HBS-EP buffer (10 mM Hepes, 0.15 M NaCl, 2.5 mM EDTA and 0.005%Tween-20) at 25° C. Binding of the His-FBG variants to the immobilisedB12-hIgG4 was also measured in HBS-EP buffer at 25° C. The flow rate was5 μl/min in the immobilization experiments and 20 μl/min for kineticanalyses. The sensor chip surface was regenerated using 3 M MgCl₂. Datawere analysed using BIAevaluation program 4.1 (GE Healthcare).

Analysis of B12-IgG4 binding to citrullinated His-FBG revealed that thekinetic parameters were essentially unchanged when compared to valuesobtained for binding to unmodified His-FBG (Table 13). These resultsindicate that anti-FBG antibodies of the B12 lineage would be expectedto bind both citrullinated and non-citrullinated forms of TNC FBG intherapeutic or diagnostic applications.

TABLE 13 Kinetic parameters for interaction of B12-hIgG4 with theHis-FBG variants. Each kinetic parameter represents the mean ± s.d. of 3independent determinations. Analyte K_(D)(M) K_(on) (M⁻¹s⁻¹)K_(Off)(s⁻¹) His-FBG (1.7 ± 0.3) × 10⁻¹⁰ (4.1 ± 0.6) × 10⁶ (6.8 ± 0.9) ×10⁻⁴ His-FBG + PAD2 (3.2 ± 0.3) × 10⁻¹⁰ (3.0 ± 0.4) × 10⁶ (9.6 ± 0.8) ×10⁻⁴ His-FBG + PAD4 (3.2 ± 0.7) × 10⁻¹⁰ (2.6 ± 0.6) × 10⁶ (7.8 ± 0.4) ×10⁻⁴

Example 8—Detection of TNC FBG in Human RA Tissue UsingImmunohistochemistry

Immunohistochemistry studies were performed to determine whetheranti-FBG antibodies effectively recognise endogenous forms of the humanTNC FBG protein in human tissue. Tenascin-C is expressed at sites ofchronic inflammation and its localisation within the inflamed synoviumof joints from individuals with rheumatoid arthritis has previously beendemonstrated by immunohistochemistry using commercially availableantibodies (Goh et al, 2010; Salter D M, 1993).

The B12 antibody was expressed as mouse IgG2a format using the QMCFexpression technology (Icosagen, Estonia) and purified by Protein Gaffinity chromatography followed by Superdex 200 gel filtration. Controlmouse IgG1 anti-tenascin-C antibody (Clone 4F10TT; Takara Clontech),which recognises an EGF domain of full-length human tenascin-C was usedas a positive control comparator. Mouse IgG1 (Dako X0931) or IgG2a (DakoX0943) against an irrelevant bacterial antigen were used as controlprimary antibodies to determine the level of non-specific backgroundstaining with these isotypes. Frozen sections of human knee jointsynovium from donors with confirmed RA diagnosis (Asterand, UK) wereequilibrated to room temperature, fixed (10 min) in 1:1 v/vacetone/methanol, and transferred to wash buffer. Immunostaining wasperformed using a Dako Autostainer with Envision Flex reagents (DakoK8010) according to manufacturer's protocols. Briefly, fixed tissueslides were placed onto the automated stainer and blocked (peroxidaseblock, 5 min; protein block, 10 min, Dako X0909) before 30 minapplication of primary antibody (B12 or Clone 4F10TT; 1, 2, or 4 μg/ml).In some controls, slides were not exposed to primary antibody. Afterwashing, HRP-labelled goat anti-mouse secondary antibody was applied (20min) and slides were washed again, followed by 10 min application ofDAB+ Chromogen. Slides were washed, counterstained with haematoxylin andcoverslipped for microscopic visualisation of staining.

In cryosections of RA synovium that were fixed using acetone/methanol,the anti-TNC FBG B12 mouse IgG2a showed a very similar pattern ofstaining to that obtained with the positive control antibody Clone4F10TT. Specific immunostaining was observed in the synovium, fibrouscapsule, vasculature and within the interstitium. There was no stainingwithin lymphoid aggregates (FIGS. 13A, 13C). Some non-specificimmunostaining was present in non-immune control treated tissues (FIGS.13B, 13D). These results confirm and extend previous reports oftenascin-C expression within RA synovium, demonstrating that B12 is aneffective agent for binding endogenous tenascin-C at sites ofinflammation and further indicating that FBG is an accessible target inRA.

Example 9—Antibody Sequences

Antibody 2A5 VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

or

Antibody B12 VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)VH CDR3: DISAVPDTFDI (SEQ ID NO: 11) VH amino acid sequence:

S (SEQ ID NO: 12) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody D8 VH CDR1: SYGIS (SEQ ID NO: 16)VH CDR2: WISAYNGNTNYAQKLQG (SEQ ID NO: 17)VH CDR3: NQDSSSDY (SEQ ID NO: 18) VH amino acid sequence:

(SEQ ID NO: 19) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTLQT (SEQ ID NO: 20)VL amino acid sequence:

Antibody F3 VH CDR1: SYGMH (SEQ ID NO: 22)VH CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 23)VH CDR3: EGYDQLFSAESNAFDI (SEQ ID NO: 24) VH amino acid sequence:

LVTVSS (SEQ ID NO: 25) VL CDR1: TRSSGSIASYFVQ (SEQ ID NO: 26)VL CDR2: EDNQRPS (SEQ ID NO: 27) VL CDR3: QSYDSSNWV (SEQ ID NO: 28)VL amino acid sequence:

NO: 29); or

127) Antibody 165 13 B1 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

SS (SEQ ID NO: 31) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 165 13 B6 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

SS (SEQ ID NO: 33) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 165 13 D1 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

SS (SEQ ID NO: 35) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 165 13 C3 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

SS (SEQ ID NO: 37) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 165 13 D4 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

SS (SEQ ID NO: 39) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 165 13 A4 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

S (SEQ ID NO: 41) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 165 13 B3 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

SS (SEQ ID NO: 43) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 165 13 E1 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

SS (SEQ ID NO: 45) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 180 11 F5 (derived from B12) VH CDR1: DYAMH (SEQ ID NO: 9)VH CDR2: GISGSGGSTYYADSVKG (SEQ ID NO: 10)

VH amino acid sequence:

SS (SEQ ID NO: 47) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: DASNLET (SEQ ID NO: 13) VL CDR3: QQSYSTPQT (SEQ ID NO: 14)VL amino acid sequence:

Antibody 160 01 E3 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

S (SEQ ID NO: 49) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 160 01 D6 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 20

VH amino acid sequence:

(SEQ ID NO: 51) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 160 01 H4 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

S (SEQ ID NO: 53) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 160 01 A4 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

(SEQ ID NO: 55) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 160 01 F1 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

S (SEQ ID NO: 57) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 160 01 G2 (derived form 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

(SEQ ID NO: 59) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 161 01 F6, also known as 160 01 F6 (derived from 2A5)VH CDR1: ELSMH (SEQ ID NO: 1) VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

(SEQ ID NO: 61) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 161 01 A12 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

(SEQ ID NO: 63) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 161 01 C09 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

(SEQ ID NO: 65) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 161 01 H10 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

S (SEQ ID NO: 67) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 161 01 C11 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

S (SEQ ID NO: 69) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 162 02 D3 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)

VH amino acid sequence:

(SEQ ID NO: 71) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: QQSYSTPWT (SEQ ID NO: 7)VL amino acid sequence:

Antibody 162 02 C6 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

Antibody 162 02 H5 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 50VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

Antibody 162 02 F3 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

Antibody 162 02 C1 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

Antibody 162 02 C2 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

Antibody 162 02 F4 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

Antibody 162 02 C3 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

 

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

Antibody 162 02 E11 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

or

Antibody 163 02 A12 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6)

VL amino acid sequence:

Antibody 163 02 D11 (derived from 2A5) VH CDR1: ELSMH (SEQ ID NO: 1)VH CDR2: GFDPEDGETIYAQKFQG (SEQ ID NO: 2)VH CDR3: AQKETYALTY (SEQ ID NO: 3) VH amino acid sequence:

(SEQ ID NO: 4) VL CDR1: RASQYIQGFLN (SEQ ID NO: 5)VL CDR2: AASTLQD (SEQ ID NO: 6) VL CDR3: LHHYKSPWT (SEQ ID NO: 90)VL amino acid sequence:

IgG4 165 13 C3 (constant region with hinge modification as described inAngal 1993)Reference: Angal S1, King D J, Bodmer M W, Turner A, Lawson A D, Roberts G,Pedley B, Adair J R. Mol Immunol. 1993 Jan;30(1):105-8.QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKSYQSDEDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHTYQKSLSLSLGK

Example 10—Protein Sequences

Amino acid sequence of human tenascin-C FBG domain [SEQ ID NO: 92]IGLLYPFPKDCSQAMLNGDTTSGLYTIYLNGDKAEALEVFCDMTSDGGGWIVFLRRKNGRENFYQNWKAYAAGFGDRREEFWLGLDNLNKITAQGQYELRVDLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALSYKGAFWYRNCHRVNLMGRYGDNNHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRNLEGRRKRAAmino acid sequence of mouse tenascin-C FBG domain [SEQ ID NO: 93]IGLLYPFPRDCSQAMLNGDTTSGLYTIYINGDKTQALEVYCDMTSDGGGWIVFLRRKNGREDFYRNWKAYAAGFGDRREEFWLGLDNLSKITAQGQYELRVDLQDHGESAYAVYDRFSVGDAKSRYKLKVEGYSGTAGDSMNYHNGRSFSTYDKDTDSAITNCALSYKGAFWYKNCHRVNLMGRYGDNNHSQGVNWFHWKGHEYSIQFAEMKLRPSNFRNLEGRRKRAAmino acid sequence of rat tenascin-C FBG domain [SEQ ID NO: 94]IGLLYPFPRDCSQAMLNGDTTSGLYTIYINGDKTQALEVYCDMTSDGGGWIVFLRRKNGREDFYRNWKAYATGFGDRREEFWLGLDNLSKITAQGQYELRVDLQDHGESAYAVYDRFSVGDAKSRYKLKVEGYSGTAGDSMNYHNGRSFSTYDKDTDSAITNCALSYKGAFWYKNCHRVNLMGRYGDNNHSQGVNWFHWKGHEYSIQFAEMKLRPSNFRNLEGRRKRAAmino acid sequence of dog tenascin-C FBG domain [SEQ ID NO: 95]IGLLYPFPRDCSQAMLNGDTTSGLYTIYLNGDKAQALEVYCDMTSDGGGWIVFLRRKNGREDFYRNWKAYAAGFGDRREEFWLGLDNLHKITAQGQYELRVDLRDHGKTAYAVYDRFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALSYKGAFWYKNCHRVNLMGRYGDNNHSQGVNWFHWKGHEYSIQFAEMKLRPSNFRNLEGRRKRAAmino acid sequence of human tenascin-R FBG domain [SEQ ID NO: 96]FPHPQDCAQHLMNGDTLSGVYPIFLNGELSQKLQVYCDMTTDGGGWIVFQRRQNGQTDFFRKWADYRVGFGNVEDEFWLGLDNIHRITSQGRYELRVDMRDGQEAAFASYDRFSVEDSRNLYKLRIGSYNGTAGDSLSYHQGRPFSTEDRDNDVAVTNCAMSYKGAWWYKNCHRTNLNGKYGESRHSQGINWYHWKGHEFSIPFVEMKMRPYNHRLMAGRKRQSLQF

Example 11—Germlined Sequences

Closest germline matches were determined using IMGT/DomainGapAlign:Ehrenmann F., Kaas Q. and Lefranc M. P. Nucleic Acids Res., 38, D301-307(2010)

Changes from non-germlined sequences are shown by an underline of theamino acid. The CDRs are shown by the boxed sequences.

Antibody 2A5 Framework Germlined: VH amino acid sequence:(SEQ ID NO: 112)

Framework Germlined: VL amino acid sequence: (SEQ ID NO: 113)

or (SEQ ID NO: 139)

CDRs changed as a result of the germlined sequence: VL CDR2:  (SEQ ID NO: 114) AASSLQS Antibody B12Framework Germlined: VH amino acid sequence: (SEQ ID NO: 115)

CDRs changed as a result of the germlined sequence: VH CDR2:  (SEQ ID NO: 116) GISGSGGSTYYADSVKYFramework Germlined: VL amino acid sequence: (SEQ ID NO: 117)

or (SEQ ID NO: 140)

CDRs changed as a result of the germlined sequence: VL CDR2:  (SEQ ID NO: 118) DASSLQS Antibody D8Framework Germlined: VH amino acid sequence: (SEQ ID NO: 119)

Framework Germlined: VL amino acid sequence: (SEQ ID NO: 120)

or (SEQ ID NO: 141)

CDRs changed as a result of the germlined sequence: VL CDR2:  (SEQ ID NO: 121) DASSLQS Antibody F3Framework germlined: VH amino acid sequence: (SEQ ID NO: 122)

Framework germlined: VL amino acid sequence: (SEQ ID NO: 123)

or  (SEQ ID NO: 142)

Example 12—Use of Antibodies in Western Blotting

In order to confirm that the monoclonal antibodies IgG4 C3 (165_13_03 asreferred to above) and IgG4 B12 could be successfully used for westernblotting, first specificity was tested using purified proteins (FIGS.14A-14D). Next, glioma cell lysate was used to determine the ability ofB12 to detect full length TNC in a biologically relevant mixture ofother proteins (FIGS. 15A-15B).

As shown by the data in FIGS. 14A-14D, recombinant CD4-TNC-FBG(Nascient), CD4-TNR-FBG or FIBRINOGEN (KIR) were run on four 10%SDS-PAGE gels under reducing conditions before blotting tonitrocellulose membranes and detection with the following antibodies A)C3 IgG4 MAb (Nascient) at 1:20,000 (0.25 ug/ml), overnight at 4° C. B)B12 IgG4 MAb (Nascient) at 1:20,000 (0.25 ug/ml), overnight at 4° C. C)Anti-Tenascin-R antibody (Santa Cruz Biotechnology, sc-9875) at 1:2,000(0.1 ug/ml) overnight at 4° C. D) Anti-TNC-FBG polyclonal antibody(Midwood group) at 1:500, overnight at 4° C. The secondary antibody usedfor C3 and B12 antibodies was Abcam (ab6858) at 1:20,000 for 1 h at RT.For TNR antibody the secondary was HRP conjugated anti-goat(Sigma-Aldrich, SAB3700259) at 1:10,000 for 1 h at RT. For thepolyclonal TNC antibody the secondary used was HRP conjugatedanti-Rabbit (DAKO, P0217) at 1:5,000 for 1 h at RT. Exposure to film was5 min for all blots shown.

In this experiment both C3 and B12 showed specificity for TNC-FBG withvery little cross-reactivity with either TNR-FBG or Fibrinogen,indicating their suitability for western blotting applications as theyshow good specificity for TNC-FBG.

As shown by the data in FIGS. 15A-15B, glioma cell lysate (KIR) andtenascin-C(Nascient) were run on a 5% SDS-PAGE gel under reducingconditions before blotting to membranes and detection with A. B12 IgG4Mab (Nascient) at 1:20,000, overnight at 4° C.; B. IgG4 isotype control(Eureka therapeutics) at 1:4,000, overnight at 4° C. The secondaryantibody used was Abcam (ab6858) at 1:10,000 for 1 h at RT. Blots weredeveloped with ECL Western Blotting Detection Reagent (GE Healthcare,Amersham). Blots were exposed to film for 1 minute.

These results indicate that B12 can detect full length TNC as well asbreakdown products and/or splice variants of TNC, and shows lowcross-reactivity to other proteins present in the cell lysate.

Example 13—Activity of the C3 Antibody In Vitro

In order to confirm that the monoclonal antibody C3 (165_13_C3) acts bydisrupting the binding of TNC-FBG to its receptor TLR4, first an invitro binding assay was developed for TLR4 and Fc-His-FBG then theeffect of pre-incubation of Fc-His-FBG with C3 was determined.

Recombinant human TLR4 (R&D systems) (1 ug/ml (14.6 nM)) in PBS (or PBSalone) was bound to a 96-well plate. After blocking (10% BSA) theindicated concentrations of Human Fc-His-FBG was added and detection wascarried out by incubation of an anti-human IgG1 MAb (AbD Serotec, clone2011) at 1 ug/ml, an anti-mouse HRP conjugated secondary antibody (AbDSerotec, STAR13B) at 1 ug/ml, and TMB substrate. The results are shownin FIG. 16A, n=4 mean and SEM shown. This experiment shows thatFc-His-FBG binds TLR4 in vitro in a dose dependent manner.

As shown in FIG. 16B, monoclonal Ab C3 disrupts the binding FBG and TLR4in vitro. Recombinant human TLR4 in PBS (or PBS alone) was bound to a96-well plate, after blocking recombinant human Fc-His-TNC-FBG (100 nM)which had been pre-incubated with C3 Mab or isotype control antibody wasadded. Detection was carried out by successive incubation of antibodydirected against the Fc portion of the protein, an anti-mouse HRPconjugated secondary antibody and TMB substrate. The percentageinhibition in the C3 pre-incubated samples was calculated compared tothe isotype control samples (IC50=44.5 nM). n=4.

Example 14—Anti-Inflammatory Effect of Antibodies B12, A4 and C3

It was confirmed that the anti-TNC-FBG antibodies B12, A4 (160_01_A4)and C3 (165_13_03) have an anti-inflammatory effect in a biologicalsystem. To do this, human monocytes were isolated from peripheral blood(London blood bank) by Ficoll gradient and counter-flow centrifugation.The monocytes were then differentiated with 100 ng/ml M-CSF (Peprotec)for 5 days to produce M2 macrophages.

As shown by the results in FIG. 17A, recombinant human Fc-TNC FBG (1 uM)or LPS (Enzo) (1 ng/ml) was pre-incubated for 30 min at RT with MAb C3(1, 0.2, and 0.04 uM) or isotype control (Eureka) MAb (1 uM) beforebeing added in triplicate to Human M2 macrophage cultures. After 24 hsupernatants were taken and subjected to IL-8, IL-6 and TNF cytokineELISA (BD Biosciences), n=3. These results show that at 1 uM C3 greatlyreduces the pro-inflammatory cytokine release by human M2 macrophagesstimulated with TNC-FBG, this reduction is statistically significant forboth IL-8 and TNF. As expected C3 has no effect on LPS-induced cytokinerelease.

FIG. 17B shows results from the experiment where recombinant murineFc-TNC FBG (1 uM) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2and 0.04 uM) or isotype control MAb (Eureka) (1 uM) before being addedin triplicate to Human M2 macrophage cultures. After 24 h supernatantswere taken and subjected to cytokine ELISA. n=3 or over, mean and SEMshown. Again C3 at 1 uM greatly reduced the murine Fc-TNC-FBG-inducedcytokine release by macrophages, indicating good cross-speciesreactivity of the antibody. To confirm that the FBG-induced cytokinerelease was induced by the FBG rather than the Fc portion of theprotein, a protein where the Fc portion is mutated to be inactive(Fc-Mut-FBG) was used, Anti-TNC-FBG antibodies, B12, C3 (165_13_03) andA4 (160_01_A4) were also tested for activity against this molecule.Fc-Mut-FBG (1 uM) and C3, A4 or B12 (1 uM) were pre-incubated for 30 minat RT before being added to human M2 macrophage cultures. After 24 hsupernatants were taken and subjected to cytokine ELISA. n=3, mean andSEM shown. Results are shown in FIG. 17C. This experiment confirms thatFc-His-FBG-induced cytokine synthesis is not due to the Fc portionsignalling through Fc-receptors. Further, it shows that pre-incubationof the related antibodies B12 and A4, as well as C3 greatly reduceFBG-induced cytokine release by human M2 macrophages.

FIG. 18A shows that Monoclonal antibody B12 reduces the production ofpro-inflammatory cytokines by primary human macrophages stimulated withhuman TNC-FBG. In that experiment, recombinant Human tenascin-C FBG (1uM) was pre-incubated for 30 min at RT with MAb B12 (1, 0.1, 0.01 or0.001 uM) or isotype control MAb (1 uM) before being added in triplicateto Human M2 macrophage cultures. After 24 h supernatants were taken andsubjected to cytokine ELISA, n=1. Here again we see that the B12antibody pre-incubation reduces FBG-induced cytokine release, in thisdonor IL-8 gives a minimal response.

FIG. 18B shows that monoclonal antibody C3 produced at laboratory orlarger scale show the same level of efficacy in blockade of FBG-inducedcytokine synthesis by primary human macrophages.

To take the C3 antibody into animal studies, IgG4 B12 165-13-C3 productwas cloned, expressed and purified at a leading contract manufacturingorganisation using a commercial GS-CHO expression. cDNAs for the heavyand light chain variable regions were optimised for CHO expression andsynthesised (with commercial signal sequences) by Life Technologiesprior to cloning into the expression vectors. CHO cells were transfectedas pools and the highest expressing pool was taken forward intolarge-scale shake flask production (22 L-11×2 L in 5 L shake flasks.).Proprietary feeds were administered on day 4 and 8 prior to harvestingthe culture on day 12. Material was centrifuged prior to depthfiltration and filter sterilisation. Approximately a 5.5 foldconcentration of material was performed using tangential flow filtration(30 kDa molecular weight cut off) and the resulting concentrate wasfilter sterilised again prior to MabSelect SuRe purification. Theproduct was eluted and product was neutralised and thenconcentrated/diafiltered to approximately 11 mg/mL in 20 mM NaOAc, pH5.5, 150 mM NaCl. Reduced and non-reduced SDS-PAGE analysis togetherwith size exclusion—HPLC showed material that was highly pure andgreater than 98% monomer. Endotoxin was less than 0.1 Eu per mg.

In this experiment the potency of the larger scale antibody batch wascompared to the current smaller scale batch. Recombinant Humantenascin-C FBG (1 uM) was pre-incubated for 30 min at RT with MAb C3 (1,0.2 and 0.04 uM) or isotype control MAb (1 uM) before being added intriplicate to Human M2 macrophage cultures. After 24 h supernatants weretaken and subjected to cytokine ELISA. n=1, Ico=laboratory scaleLon=larger scale material. This experiment shows that both batches ofantibodies show equal potency in the reduction of FBG-induced cytokinesynthesis, i.e. the results are consistent irrespective of production.

Example 15—Monoclonal Antibody C3 (165 13 C3) Reduces the Production ofPro-Inflammatory Cytokines by RA Synovial Fibroblasts Stimulated withHuman TNC-FBG

It has been reported that synovial fibroblasts could be an importantsource of pro-inflammatory cytokine release in RA (R Bucala et al.(1991) Constitutive Production of Mitogenic and Inflammatory Cytokinesby Rheumatoid Synovial Fibroblasts. J. Exp. Med. 173:569-574), it wastherefore tested whether the C3 antibody also showed similar effects onFBG-induced cytokine release as in the macrophages.

Human RA fibroblasts were grown out of donor RA synovial tissue bydigestion of the tissue in RPMI (Lonza) containing 0.5 mg/ml Liberase(Roche) and 0.2 mg/ml DNase (Roche) and incubation at 37° C. for 1-1.5h. The resulting tissue was pipetted through a 200 μm nylon mesh; thematerial that did not pass through the mesh was put into a petri-dishcontaining RPMI with 10% FBS (Life technologies) and 1% pen/strep (Lifetechnologies) and incubated at 37° C. for 5 days. After 5 days synovialfibroblasts grow out of the tissue and the remaining tissue was removedfrom the RA synovial fibroblast (RASF) culture which was subsequentlymaintained in DMEM (Lonza) containing 10% FBS and 1% pen/strep. For thisexperiment RASF were plated out at 1×10⁴ cells/well. Recombinant HumanTNC-FBG (1 uM) was pre-incubated for 30 min at RT with MAb C3 (1, 0.2and 0.04 uM) or isotype control MAb (1 uM) before being added intriplicate to the synovial fibroblast cultures. After 24 h supernatantswere taken and subjected to cytokine ELISA. n=1, mean and SEM shown (seeFIG. 19). These results indicate that C3 acts to reduce FBG inducedpro-inflammatory cytokine release (both IL-8 and IL-6) in RA synovialfibroblasts, showing that this is a potential mechanism in multiple celltypes found in the inflamed RA joint.

Example 16—Levels of Tenascin-C in Rat Model

Expression of tenascin-C in both mouse and rat CIA (collagen-inducedarthritis) models was confirmed and disease activity shown to correlatewith clinical score.

FIG. 20 shows the results of an experiment measuring the levels oftenascin-C in synovial fluid wash-out from the paws of rats at theconclusion of two separate CIA studies (KWS). Tenascin-C levels weremeasured by ELISA (IBL, large (FN III-B) kit). The measured TNC levelwas then correlated with the clinical score associated with that pawdesignated by KWS. This experiment shows that the higher the clinicalscore for the paw, the higher the level of TNC seen in the synovialfluid from that paw. This indicates that the rat CIA model is a goodmodel for testing of the C3 antibody.

Example 17—Evaluation of C3 Antibody in a Rat Model of Collagen-InducedArthritis

IgG4 C3 (165_13_03) was tested for therapeutic activity in the standardrat collagen induced arthritis model. Adult male Lewis rats wererandomly allocated to experimental groups and allowed to acclimatise forone week. On Day 0, animals were administered with 500 μl of a 1 mg/mlemulsion of type II bovine collagen in incomplete Freund's adjuvant(CII/IFA) by intra-dermal injection in the lower back. On Day 7, animalsreceived a second injection of CII/IFA. Injections were performed undergas (isoflurane) anaesthesia. Treatments were administered according tothe Administration Schedule shown below in Table 14.

TABLE 14 Administration Schedule Disease Group Treatment Dose RouteRegimen Induction 1 Vehicle (0.9% NaCl) n/a IV Twice Day 0, Day 2Control IgG4 ¹ 10 mg/kg IV weekly*, 7: CII/IFA, 3 IgG4 165_13_C3 1 mg/kgIV Day 0-End ID 4 IgG4 165_13_C3 3 mg/kg IV 5 IgG4 165_13_C3 10 mg/kg IV¹ Fully human IgG4 isotype control, preclinical grade, (ET904, EurekaTherapeutics), n/a: not applicable, IV: intra-venous injections, ID:intra-dermal injections, CII/IFA: Type II collagen and IncompleteFreund's Adjuvant emulsion, *Day 0, Day 3, Day 7, Day 10, Day 14, Day17, Day 21 and Day 24

From Day 7 until the end of the experiment, animals were scored threetimes per week for clinical signs of arthritis by an experimenter blindto the treatments. On Day 0, Day 14, Day 21 and Day 28, paw volumes weremeasured using a plethysmometer by an experimenter blind to thetreatments.

Results

Non-Specific Clinical Observations

From Day 0 until the end of the experiment, animals were checked dailyfor non-specific clinical signs to include abnormal posture (hunched),abnormal coat condition (piloerection) and abnormal activity levels(reduced or increased activity). One animal in Group 6 (ID #6.9,antibody 10 mg/kg-treated) did not recover from the isofluraneanaesthesia on Day 21. Animals did not show any non-specific clinicalsigns such as abnormal posture, abnormal coat condition and abnormalactivity levels. One animal in Group 1 (ID #1.10, vehicle-treated) wasculled on Day 22, prior to the end of the experiment, due to theseverity of the clinical signs of arthritis.

Clinical Scores

From Day 7 until the end of the experiment, animals were scored threetimes per week for clinical signs of arthritis to include front and hindlimb swelling. The experimenter was blind to the treatments. Each limbwas scored on a five-point scale: (0) absence of swelling, (1) slightswelling and/or erythema, (2) mild swelling, (3) moderate swelling and(4) severe swelling and/or joint rigidity. A clinical score wascalculated for each animal by adding the score of each limb. Dataprovided in FIG. 21 were graphed (Mean±SEM for each experimental group)and analysed by two-way ANOVA followed by Dunnett's post-test formultiple comparisons between experimental groups. The last recordedscore for the vehicle-treated animal #1.10 was used after Day 22. Datarecorded from animal #6.9 were excluded from the analysis. Clinicalscores in the vehicle-treated group significantly increased from Day 17until the end of the experiment on Day 28 when compared to the clinicalscores measured on Day 7 (p<0.0001). Control IgG4 and IgG4 C3 1 mg/mLdose groups did not induce any significant difference when compared tothe vehicle-treated group between Day 7 and the end of the experiment onDay 28. IgG4 C3 administered at 3 mg/kg, induced a significant reductionof the clinical scores when compared to the vehicle-treated group on Day24 (p<0.01). IgG4 C3 administered at 10 mg/kg, induced a significantreduction of the clinical scores when compared to the vehicle-treatedgroup from Day 22 until the end of the experiment on Day 28 (p<0.01).

Paw Volumes

On Day 0, Day 14, Day 21 and Day 28, hind paw volumes were measuredusing a plethysmometer (water-displacement device). Measurements wereperformed under gas (isoflurane) anaesthesia. The experimenter was blindto the treatment. Right and left hind paw volumes from each animal oneach experimental day were averaged. FIG. 22 shows graphed data(Mean±SEM for each experimental group). Data were analysed by two-wayANOVA followed by Dunnett's post-test for multiple comparisons betweenexperimental groups. The last recorded value for the vehicle-treatedanimal #1.10 was used on Day 28. Data recorded from animal #6.9 wereexcluded from the analysis.

Paw volumes measured in the vehicle-treated group increasedsignificantly from Day 14 until the end of the experiment on Day 28 whencompared to the paw volumes measured on Day 0 (p<0.01 on Day 14,p<0.0001 on Day 21 and Day 28). The control IgG4 and 1 mg/kg IgG4 C3dose groups did not induced any difference in hind paw volumes whencompared to the vehicle-treated group between Day 0 and Day 28. IgG4 C3administered at 3 mg/kg induced a significant decrease of the hind pawvolumes when compared to the vehicle-treated group on Day 28 (p<0.01).IgG4 C3 administered at 10 mg/kg induced a significant decrease of thehind paw volumes when compared to the vehicle-treated group on Day 21(p<0.05) and Day 28 (p<0.01).

CONCLUSIONS

The test antibody, IgG4 C3 (165_13_03), when administered at 3 mg/kg or10 mg/kg, significantly reduced the severity of the clinical signs.

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Embodiments of the invention will now be described in the followingnumbered paragraphs:

1. An antibody or antigen-binding fragment, derivative or variantthereof which is capable of binding to the FBG domain of tenascin-C,wherein the antibody or antigen-binding fragment, derivative or variantthereof comprises: one or more sequences selected from SEQ ID NOs: 1-8,48-91 and 112-114; and/or one or more sequences selected from SEQ IDNOs: 5, 9-15, 30-47 and 115-118; and/or one or more sequences selectedfrom SEQ ID NOs: 5, 13, 16-21 and 119-121; and/or one or more sequencesselected from SEQ ID NOs: 22-29 and 122-123.

2. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 wherein the antibody or antigen-binding fragment,derivative or variant thereof comprises: one or more CDR sequencesselected from SEQ ID NOs: 1-3, 5-7, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 and 114; and/or oneor more CDR sequences selected from SEQ ID NOs: 9-11, 5, 13-14, 30, 32,34, 36, 38, 40, 42, 44, 46, 116 and 118; and/or one or more CDRsequences selected from SEQ ID NOs 16-18, 5, 13, 20 and 121; and/or oneor more CDR sequences selected from SEQ ID NOs 22-24 and 26-28.

3. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 or 2 wherein the antibody or antigen-bindingfragment, derivative or variant thereof comprises: one or more CDR3sequences selected from SEQ ID NOs: 3, 7, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90; and/orone or more CDR3 sequences selected from SEQ ID NOs: 11, 14, 30, 32, 34,36, 38, 40, 42, 44 and 46; and/or one or more CDR3 sequences selectedfrom SEQ ID NOs 18 and 20; and/or one or more CDR3 sequences selectedfrom SEQ ID NOs 24 and 28.

4. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 3 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises: oneor more CDR3 sequences selected from SEQ ID NOs: 3, 54, 66 and 70;and/or one or more CDR3 sequences selected from SEQ ID NOs: 7, 76, 88and 90; and/or one or more CDR3 sequences selected from SEQ ID NOs: 11,30, 34 and 36.

5. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 1 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises: a VHCDR3 sequence selected from SEQ ID NOs: 3, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68 and 70; a VH CDR3 sequence selected from SEQ ID NOs: 3,54, 66 and 70; or a VH CDR3 sequence selected from SEQ ID NOs: 3 and 54.

6. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 1 or 5 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises: a VLCDR3 sequence selected from SEQ ID NOs: 7, 72, 74, 76, 78, 80, 82, 84,86, 88 and 90; a VL CDR3 sequence selected from SEQ ID NOs: 7, 76, 88and 90; or a VL CDR3 sequence of SEQ ID NO 7.

7. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 1 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises a VHsequence comprising the sequence of SEQ ID NO: 4 or 112, and wherein theVH sequence comprises a CDR3 sequence which is replaced with: a VH CDR3sequence selected from SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68 and 70; a VH CDR3 sequence selected from SEQ ID NOs: 54, 66 and70; or a VH CDR3 sequence of SEQ ID NO: 54.

8. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 1 or 7 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises a VLsequence comprising the sequence of SEQ ID NO: 8 or 113, and wherein theVL sequence comprises a CDR3 sequence which is replaced with: a VL CDR3sequence selected from SEQ ID NOs: 72, 74, 76, 78, 80, 82, 84, 86, 88and 90; or a VL CDR3 sequence selected from SEQ ID NOs: 76, 88 and 90.

9. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 1 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises: a VHCDR3 sequence selected from SEQ ID NOs: 11, 30, 32, 34, 36, 38, 40, 42,44 and 46; a VH CDR3 sequence selected from SEQ ID NOs: 11, 30, 34 and36; or a VH CDR3 sequence selected from SEQ ID NOs 11, 30 and 36.

10. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 1 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises a VLsequence comprising the sequence of SEQ ID NO: 15 or 117.

11. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 1 or 10 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises a VHsequence comprising the sequence of SEQ ID NO: 12 or 115, and whereinthe VH sequence comprises a CDR3 sequence which is replaced with: a VHCDR3 sequence selected from SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44and 46; a VH CDR3 sequence selected from SEQ ID NOs: 30, 34 and 36; or aVH CDR3 sequence selected from SEQ ID NOs 30 and 36.

12. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 wherein the antibody or antigen-binding fragment,derivative or variant thereof comprises: a VL CDR3 sequence of SEQ IDNO: 7 and a VH CDR3 sequence selected from SEQ ID NOs: 3 and 48-70; orcomprises a VH CDR3 sequence of SEQ ID NO: 3 and a VL CDR3 sequenceselected from SEQ ID NOs: 7 and 72-90; or comprises a VL CDR3 sequenceof SEQ ID NO: 14 and a VH CDR3 sequence selected from SEQ ID NOs: 11 and30-46; or comprises a VH CDR3 sequence of SEQ ID NO: 18 and a VL CDR3sequence of SEQ ID NO: 20; or comprises a VH CDR3 sequence of SEQ ID NO:24 and a VL CDR3 sequence of SEQ ID NO: 28.

13. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 wherein the antibody or antigen-binding fragment,derivative or variant thereof comprises: at least one CDR sequenceselected from SEQ ID NOs: 1-3, and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 48 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 50 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 52 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 54 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 56 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 58 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 60 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 62 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 64 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 66 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 68 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1, 2, 70 and 5-7; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 72; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5-6 and 74; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 76; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 78; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 80; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 82; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 84; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 86; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 88; or at least one CDR sequenceselected from SEQ ID NOs: 1-3, 5, 6 and 90; or at least one CDR selectedfrom SEQ ID NOs: 1-3, 5, 7 and 114; or at least one CDR sequenceselected from SEQ ID NOs: 9-11 and 5, 13 and 14; or at least one CDRsequence selected from SEQ ID NOs: 9, 10, 30, 5, 13 and 14; or at leastone CDR sequence selected from SEQ ID NOs: 9, 10, 32, 5, 13 and 14; orat least one CDR sequence selected from SEQ ID NOs: 9, 10, 34, 5, 13 and14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10, 36, 5,13 and 14; or at least one CDR sequence selected from SEQ ID NOs: 9, 10,38, 5, 13 and 14; or at least one CDR sequence selected from SEQ ID NOs:9, 10, 40, 5, 13 and 14; or at least one CDR sequence selected from SEQID NOs: 9, 10, 42, 5, 13 and 14; or at least one CDR sequence selectedfrom SEQ ID NOs: 9, 10, 44, 5, 13 and 14; or at least one CDR sequenceselected from SEQ ID NOs: 9, 10, 46, 5, 13 and 14; or at least one CDRselected from SEQ ID NOs: 9, 11, 116, 5, 14 and 118; or at least one CDRsequence selected from SEQ ID NOs: 16-18 and 5, 13 and 20; or at leastone CDR sequence selected from SEQ ID NOs: 16-18 and 5, 121 and 20; orat least one CDR sequence selected from SEQ ID NOs: 22-24 and 26-28.

14. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in in paragraph 3 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises: a VHCDR3 sequence selected from SEQ ID NOs: 3 and 54; or a VH CDR3 sequenceselected from SEQ ID NOs: 11, 30 and 36.

15. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 1 wherein the antibody orantigen-binding fragment, derivative or variant thereof comprises VHand/or VL sequences comprising: one or more sequences selected from SEQID NOs: 4, 8, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 112 and 113; and/or one or moresequences selected from SEQ ID NOs 12, 15, 31, 33, 35, 37, 39, 41, 43,45, 47, 115 and 117; and/or one or more sequences selected from SEQ IDNOs 19, 21, 119 and 120; and/or one or more sequences selected from SEQID NOs 25, 29, 122 and 123.

16. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 15 wherein the VH sequence is selected from SEQ IDNOs: 4, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 and 112; and/orselected from SEQ ID NOs 12, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 115;and/or selected from SEQ ID NOs: 19 and 119; and/or selected from: SEQID NOs 25 and 122.

17. The antibody or antigen-binding fragment, derivative or variantthereof of paragraphs 15 or 16 wherein the VL sequence is selected fromSEQ ID NOs: 8, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 113; and/or isselected from SEQ ID NOs: 15 and 117; and/or is selected from SEQ IDNOs: 21 and 120; and/or is selected from SEQ ID NOs: 29 and 123.

18. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 wherein the antibody or antigen-binding fragment,derivative or variant thereof comprises both a VH and a VL sequencecomprising the sequences of a VH and VL sequence pair selected from thesequence pairs: SEQ ID NOs 4 and 8; SEQ ID NOs 49 and 8; SEQ ID NOs 51and 8; SEQ ID NOs 53 and 8; SEQ ID NOs 55 and 8; SEQ ID NOs 57 and 8;SEQ ID NOs 59 and 8; SEQ ID NOs 61 and 8; SEQ ID NOs 63 and 8; SEQ IDNOs 65 and 8; SEQ ID NOs 67 and 8; SEQ ID NOs 69 and 8; SEQ ID NOs 71and 8; SEQ ID NOs 112 and 8; SEQ ID NOs 4 and 113; SEQ ID NOs 49 and113; SEQ ID NOs 51 and 113; SEQ ID NOs 53 and 113; SEQ ID NOs 55 and113; SEQ ID NOs 57 and 113; SEQ ID NOs 59 and 113; SEQ ID NOs 61 and113; SEQ ID NOs 63 and 113; SEQ ID NOs 65 and 113; SEQ ID NOs 67 and113; SEQ ID NOs 69 and 113; SEQ ID NOs 71 and 113; SEQ ID NOs 4 and 73;SEQ ID NOs 4 and 75; SEQ ID NOs 4 and 77; SEQ ID NOs 4 and 79; SEQ IDNOs 4 and 81; SEQ ID NOs 4 and 83; SEQ ID NOs 4 and 85; SEQ ID NOs 4 and87; SEQ ID NOs 4 and 89; SEQ ID NOs and 4 and 91; SEQ ID NOs 112 and 73;SEQ ID NOs 112 and 75; SEQ ID NOs 112 and 77; SEQ ID NOs 112 and 79; SEQID NOs 112 and 81; SEQ ID NOs 112 and 83; SEQ ID NOs 112 and 85; SEQ IDNOs 112 and 87; SEQ ID NOs 112 and 89; SEQ ID NOs and 112 and 91; SEQ IDNOs and 112 and 113; or selected from the sequence pairs: SEQ ID NOs 12and 15; SEQ ID NOs 31 and 15; SEQ ID NOs 33 and 15; SEQ ID NOs 35 and15; SEQ ID NOs 37 and 15; SEQ ID NOs 39 and 15; SEQ ID NOs 41 and 15;SEQ ID NOs 43 and 15; SEQ ID NOs 45 and 15; SEQ ID NOs 47 and 15; SEQ IDNOs 115 and 15; SEQ ID NOs 12 and 117; SEQ ID NOs 31 and 117; SEQ ID NOs33 and 117; SEQ ID NOs 35 and 117; SEQ ID NOs 37 and 117; SEQ ID NOs 39and 117; SEQ ID NOs 41 and 117; SEQ ID NOs 43 and 117; SEQ ID NOs 45 and117; SEQ ID NOs 47 and 117; and SEQ ID NOs 115 and 117; or selected fromthe sequence pairs: SEQ ID NOs 19 and 21; SEQ ID NOs 19 and 120; SEQ IDNOs 119 and 21; and SEQ ID NOs 119 and 120; or selected from thesequence pairs: SEQ ID NOs 25 and 29; SEQ ID NOs 25 and 123; SEQ ID NOs122 and 29; and SEQ ID NOs 122 and 123.

19. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 comprising a VH sequence comprising a sequenceselected from SEQ ID NOs: 4, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,71 and 112.

20. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 or 19 comprising a VL sequence comprising asequence selected from SEQ ID NOs: 8, 73, 75, 77, 79, 81, 83, 85, 87,89, 91 and 113.

21. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 or 20 comprising a VH sequence comprising thesequence of SEQ ID NO: 4 or 112.

22. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 comprising a VH sequence comprising the sequenceof SEQ ID NO: 55.

23. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 comprising a VH sequence comprising a sequenceselected from SEQ ID NOs: 12, 31, 33, 35, 37, 39, 41, 43, 45, 47 and115.

24. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 comprising a VH sequence comprising the sequenceof SEQ ID NO: 12 or 115.

25. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 comprising a VH sequence comprising the sequenceof SEQ ID NO: 31.

26. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 comprising a VH sequence comprising the sequenceof SEQ ID NO: 37.

27. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 19 or 21-22 additionally comprising a VL sequencecomprising the sequence of SEQ ID NO: 8 or 113.

28. The antibody or antigen-binding fragment, derivative or variantthereof of any of paragraphs 23-26 additionally comprising a VL sequencecomprising the sequence of SEQ ID NO: 15 or 117.

29. The antibody or antigen-binding fragment, derivative or variantthereof of paragraph 1 comprising: a VH sequence comprising the sequenceof SEQ ID NO: 37; and a VL sequence comprising the sequence of SEQ IDNO: 15.

30. An antibody or antigen-binding fragment, derivative or variantthereof according to any of the preceding paragraphs wherein theantibody or antigen-binding fragment, derivative or variant thereof is apolyclonal or a monoclonal antibody or antigen-binding fragment,derivative or variant thereof.

31. An antibody or antigen-binding fragment, derivative or variantthereof according to paragraph 30 wherein the antibody orantigen-binding fragment, derivative or variant thereof is selected fromthe group consisting of Fv fragments, scFv fragments, Fab, singlevariable domains and domain antibodies.

32. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any previous paragraph wherein the antibody orantigen-binding fragment, derivative or variant thereof is humanised.

33. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any previous paragraph wherein the antibody orantigen-binding fragment, derivative or variant thereof has specificityfor tenascin-C or a domain thereof.

34. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any of paragraph 33 wherein the antibody orantigen-binding fragment, derivative or variant thereof has specificityfor the FBG domain of tenascin-C.

35. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any of paragraph 34 wherein the antibody orantigen-binding fragment, derivative or variant thereof neutralises theactivity of the FBG domain of tenascin-C.

36. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any previous paragraph wherein said tenascin-Cis citrullinated tenascin-C.

37. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 36 wherein the citrullinatedtenascin-C is citrullinated at the FBG domain.

38. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 37 wherein the citrullinatedtenascin-C is citrullinated at only the FBG domain.

39. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any previous paragraph wherein the antibody orantigen-binding fragment, derivative or variant thereof is formodulation of a chronic inflammatory response.

40. The antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 39 wherein the antibody orantigen-binding fragment, derivative or variant thereof modulates thebiological activity of tenascin-C.

41. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in paragraph 40 wherein the agent modulates thebiological activity of tenascin-C by altering the transcription,translation and/or binding properties of tenascin-C.

42. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any previous paragraph wherein the antibody orantigen-binding fragment, derivative or variant thereof down-regulatesthe biological activity of tenascin-C.

43. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any previous paragraph wherein the antibody orantigen-binding fragment, derivative or variant thereof up-regulates thebiological activity of tenascin-C.

44. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any previous paragraph wherein the antibody orantigen-binding fragment, derivative or variant thereof is an inhibitorof transcription, translation and/or the binding properties oftenascin-C.

45. An antibody or antigen-binding fragment, derivative or variantthereof as paragraphed in any previous paragraph wherein the antibody orantigen-binding fragment, derivative or variant thereof is a competitivebinding inhibitor of tenascin-C.

46. A composition comprising an antibody or antigen-binding fragment,derivative or variant thereof as defined in any of paragraphs 1-45 and apharmaceutically acceptable carrier, excipient and/or diluent.

47. A composition as paragraphed in paragraph 46 further comprising atleast one other agent.

48. A composition as paragraphed in paragraph 47 wherein the at leastone other agent is an anti-inflammatory agent, a statin, a biologicalagent (biologicals), an immunosuppressive agent, a salicylate and/or amicrobicidal agent.

49. A composition as paragraphed in paragraph 48 wherein theanti-inflammatory agent is selected from the group consistingnon-steroidal anti-inflammatories (NSAIDs), corticosteroids,disease-modifying antirheumatic drugs (DMARDs) or immunosuppressants.

50. An antibody or antigen-binding fragment, derivative or variantthereof or composition as defined in paragraphs 1-49 for use as amedicament.

51. An antibody or antigen-binding fragment, derivative or variantthereof or composition as defined in paragraphs 1-49 for use in thetreatment of a chronic inflammatory condition.

52. Use of an antibody or antigen-binding fragment, derivative orvariant thereof or composition as defined in paragraphs 1-49 in themanufacture of a medicament for the treatment or diagnosis of a chronicinflammatory condition.

53. A method of treating a chronic inflammatory condition comprisingadministering to a subject an effective amount of an antibody orantigen-binding fragment, derivative or variant thereof or compositionas defined in paragraphs 1-49.

54. An antibody or antigen-binding fragment, derivative or variantthereof or composition as defined in paragraphs 1-49 for use in thediagnosis of a chronic inflammatory condition and/or determination ofprognosis of a patient with a chronic inflammatory condition.

55. A method of diagnosing a chronic inflammatory condition and/ordetermination of prognosis of a patient with a chronic inflammatorycondition comprising detecting the presence or absence or amount of theFBG domain of tenascin-C using an antibody or antigen-binding fragment,derivative or variant thereof or composition as defined in paragraphs1-49.

56. An antibody or antigen-binding fragment, derivative or variantthereof or method as defined in paragraph 54 or 55 wherein an increasein the amount of the FBG domain of tenascin-C detected is indicative ofa chronic inflammatory condition determination and/or of prognosis of apatient with a chronic inflammatory condition.

57. The antibody or antigen-binding fragment, derivative or variantthereof or method of paragraph 56 wherein an increase of at least 50% inthe amount of FBG domain of tenascin-C detected compared to normallevels is indicative of a chronic inflammatory condition determinationand/or prognosis of a patient with a chronic inflammatory condition.

58. An antibody or antigen-binding fragment, derivative or variantthereof or composition as defined in paragraphs 1-49 for use indetermining the appropriate treatment for an individual, wherein theamount of the FBG domain of tenascin-C detected indicates theappropriate treatment for the individual.

59. A method of determining the appropriate treatment for an individualcomprising detecting the presence or absence or amount of the FBG domainof tenascin-C using an antibody or antigen-binding fragment, derivativeor variant thereof or composition as defined in paragraphs 1-49, whereinthe amount of the FBG domain of tenascin-C detected indicates theappropriate treatment for the individual.

60. The antibody or antigen-binding fragment, derivative or variantthereof or composition or method of paragraphs 58 or 59 wherein theappropriate treatment comprises the administration of an effectiveamount of an agent or composition, the agent or composition may be oneor more of: an antibody or antigen-binding fragment, derivative orvariant thereof or composition as defined in paragraphs 1-49; DMARDS(such as methotrexate); anti-TNF drug; an anti-IL17 therapy; a T-cellco-stimulation modulator (such as Orencia™—abatacept): an interleukin-6(IL-6) inhibitor (such as Actemra™—tocilizumab); an anti-CD20 antibody(such as Rituxan™—rituxumab; a B cell activating factor (such asanti-BAFF); an inhibitor of janus kinase (JAK) (such as Tofacitinib™);an inhibitor of spleen tyrosine kinase (Syk) (such as Fostamatinib™);antiTNC antibodies or antibodies to citrullinated tenascin-C domains;and/or an agent that modulates the biological activity of citrullinatedand/or non-citrullinated tenascin-C.

61. The antibody or antigen-binding fragment, derivative or variantthereof or composition method of paragraphs 58-60 wherein theappropriate treatment targets the FBG domain of tenascin-C.

62. The antibody or antigen-binding fragment, derivative or variantthereof or composition or method of paragraphs 58-61 wherein theappropriate treatment is the administration of an effective amount of anantibody or antigen-binding fragment, derivative or variant thereof, orcomposition as defined in paragraphs 1-49.

63. The antibody or antigen-binding fragment, derivative or variantthereof or composition or method of paragraphs 58-62 wherein theindividual has a chronic inflammatory condition.

64. The antibody or antigen-binding fragment, derivative or variantthereof or composition or method of paragraphs 58-63 wherein an increasein the amount of FBG domain of tenascin-C detected indicates theappropriate treatment.

65. The antibody or antigen-binding fragment, derivative or variantthereof or composition or method of paragraph 64 wherein an increase inthe amount of FBG domain of tenascin-C detected indicates that anincreased amount of the appropriate treatment is required.

66. The antibody or antigen-binding fragment, derivative or variantthereof or composition or method of paragraphs 64 or 65 wherein theincrease in the amount of FBG domain of tenascin-C detected is anincrease of at least 50% compared to normal levels of FBG domain oftenascin-C.

67. The antibody or antigen-binding fragment, derivative or variantthereof or composition or method of paragraphs 56-66 wherein the amountof FBG domain of tenascin-C is determined by the use of one or more of:immunoassays; spectrometry; western blot; ELISA; immunoprecipitation;slot or dot blot assay; isoelectric focussing; SDS-PAGE; antibodymicroarray; immunohistological staining; radio immuno assay (RIA),fluoroimmunoassay; and/or an immunoassay using an avidin-biotin and/orstreptoavidin-biotin system.

68. An antibody or antigen-binding fragment, derivative or variantthereof, composition, use or method as paragraphed in paragraphs 51-67wherein the chronic inflammatory response is associated with a conditioncharacterised by inappropriate inflammation.

69. An antibody or antigen-binding fragment, derivative or variantthereof, composition, use or method as paragraphed in paragraphs 51-67wherein the chronic inflammatory response is associated with rheumatoidarthritis (RA), autoimmune conditions, inflammatory bowel diseases(including Crohn's disease and ulcerative colitis), non-healing wounds,multiple sclerosis, cancer, atherosclerosis, sjogrens disease, diabetes,lupus erythrematosus (including systemic lupus erythrematosus), asthma,fibrotic diseases (including liver cirrhosis), pulmonary fibrosis, UVdamage, psoriasis, ankylosing spondylitis and cardiovascular disease.

70. A kit of parts comprising:

(i) an antibody or antigen-binding fragment, derivative or variantthereof or composition as defined in paragraphs 1-49;

(ii) administration means; and

(iii) instructions for their use

71. A kit of parts as paragraphed in paragraph 70 optionally comprising

(iv) at least one other agent.

72. A kit of parts for use in determining the chronic inflammatorycondition status of a subject comprising:

(i) an antibody or antigen-binding fragment, derivative or variantthereof or composition as defined in paragraphs 1-49; and

(ii) instructions for use

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the following claims.

The invention claimed is:
 1. An isolated nucleic acid molecule encodingan anti-fibrinogen-like globe (FBG) domain of a tenascin-C antibody orbinding fragment, comprising a VH region wherein CDRH1 is SEQ ID NO: 9,CDRH2 is SEQ ID NO: 10, CDRH3 is independently selected from SEQ ID NO:11, 30, 32, 34, 36, 38, 40, 42, 44, and 46, and a VL region wherein aCDRL1 is SEQ ID NO: 5, CDRL2 is SEQ ID NO: 13 and CDRL3 is SEQ ID NO:14.
 2. An isolated nucleic acid molecule according to claim 1, whereinthe CDRH3 is SEQ ID NO:
 11. 3. An isolated nucleic acid moleculeaccording to claim 2, wherein the VH region comprises SEQ ID NO:
 12. 4.An isolated nucleic acid molecule according to claim 1, wherein CDRH3 isSEQ ID NO:
 30. 5. An isolated nucleic acid molecule according to claim4, wherein the VH region comprises SEQ ID NO:
 31. 6. An isolated nucleicacid molecule according to claim 1, wherein the CDRH3 is SEQ ID NO: 32.7. An isolated nucleic acid molecule according to claim 6, wherein theVH region comprises SEQ ID NO:
 33. 8. An isolated nucleic acid moleculeaccording to claim 1, wherein the CDRH3 is SEQ ID NO:
 34. 9. An isolatednucleic acid molecule according to claim 8, wherein the VH regioncomprises SEQ ID NO:
 35. 10. An isolated nucleic acid molecule accordingto claim 1, wherein the CDRH3 is SEQ ID NO:
 36. 11. An isolated nucleicacid molecule according to claim 10, wherein the VH region comprises SEQID NO:
 37. 12. An isolated nucleic acid molecule according to claim 1,wherein the CDRH3 is SEQ ID NO:
 38. 13. An isolated nucleic acidmolecule according to claim 12, wherein the VH region comprises SEQ IDNO:
 39. 14. An isolated nucleic acid molecule according to claim 1,wherein the CDRH3 is SEQ ID NO:
 40. 15. An isolated nucleic acidmolecule according to claim 14, wherein the VH region comprises SEQ IDNO:
 41. 16. An isolated nucleic acid molecule according to claim 1,wherein the CDRH3 is SEQ ID NO:
 42. 17. An isolated nucleic acidmolecule according to claim 16, wherein the VH region comprises SEQ IDNO:
 43. 18. An isolated nucleic acid molecule according to claim 1,wherein the CDRH3 is SEQ ID NO:
 44. 19. An isolated nucleic acidmolecule according to claim 18, wherein the VH region comprises SEQ IDNO:
 45. 20. An isolated nucleic acid molecule according to claim 1,wherein the CDRH3 is SEQ ID NO:
 46. 21. An isolated nucleic acidmolecule according to claim 20, wherein the VH region comprises SEQ IDNO:
 47. 22. An isolated nucleic acid molecule according to claim 1,wherein the VL region comprises SEQ ID NO:
 15. 23. An isolated nucleicacid molecule according to claim 1, wherein the VL region comprises SEQID NO:
 125. 24. A vector comprising the nucleic acid molecule ofclaim
 1. 25. A host cell comprising the nucleic acid molecule ofclaim
 1. 26. A host cell comprising the vector of claim 24.